Studies have highlighted a growing national problem regarding the number of untested Sexual Assault Kits (SAKs). A 2011 National Institute of Justice report revealed Los Angeles alone had 10,000 untested SAKs. This backlog has fueled the need for specific and efficient testing of SAK evidence. In traditional workflows, serology tests are used to indicate the presence of a targeted bodily fluid and prioritize samples for genetic analysis. However, given the lack of sensitivity and specificity of modern serological assays, current SAK workflows often skip serological identification altogether for a “direct to DNA” approach. While these Y-Screen workflows achieve rapid screening of samples for the presence of a detectible male contributor, they do not provide any serological information. As a result, samples lack what can be critical investigative context. Improved serological capabilities with enhanced sensitivity and specificity would provide greater confidence in results for the confirmatory identification of seminal fluid. At a minimum, forensic biologists should understand the limitations associated with traditional serological approaches to seminal fluid identification when processing SAK samples. Current serological techniques based on antigen-antibody binding have exhibited both sensitivity and specificity limitations. False positive results for semen can be obtained by non-target biological fluids such as breast milk, urine, and vaginal fluid, or by non-specific binding events. This study evaluates a promising emerging technique that combines high specificity protein biomarker detection with targeted mass spectrometry. This research targeted human-specific peptide markers for seminal fluid proteins and peptide standards to perform quantification of seminal fluid peptide targets using an Agilent 6495 mass spectrometer coupled to a 1290 series liquid chromatograph. This approach has shown to be both more specific and sensitive in identifying a bodily fluid compared to current immunological based approaches. Thus, this proteomic workflow was used to evaluate authentic false positive rates of current immunochromatographic techniques for seminal fluid identification. Self-collected vaginal swabs collected from participants not engaging in barrier-free vaginal intercourse with male partners were tested using various immunochromatographic assays designed to detect both semenogelin (Sg) (RSID™-Semen) and prostate specific antigen (PSA) (ABAcard® p30 Test and SERATEC® PSA Semiquant). Similarly, three seminal fluid biomarkers (semenogelin 1, semenogelin 2, and prostate specific antigen) were used for seminal fluid identification via mass spectrometry. Any samples producing positive results on any immunochromatographic assay were evaluated to determine whether the target protein was actually present at levels above the reported sensitivity limits of the lateral flow tests. Additionally, Sperm HY-LITER™ Express was used to microscopically confirm the absence of spermatozoa in all samples producing positive immunochromatographic results. In addition to using the quantitative proteomic assay to estimate the rate of authentic false positive results associated with lateral flow assays, this research sought to establish the correlation (or lack thereof) between absolute quantitation of seminal fluid markers and the ability to successfully generate DNA profiles. Self-collected post-coital swabs from donors engaging in barrier free vaginal intercourse with male partners over varied periods of time between 1-8 days after intercourse were collected. All samples were analyzed using the quantitative seminal fluid protein mass spectrometry assay, once again targeting SgI, SgII, and PSA. Both autosomal STR profiles (GlobalFiler™) and Y-STR profiles (Yfiler™ Plus) were subsequently generated. With regard to immunochromatographic assay false positive rates, a total of 17 false positives for semen were observed (n=150), 14 of which were consistent with PSA and 3 with Sg, for a corresponding total false positive rate of 9.3% and 2%, respectively (11.3% overall). These samples were all confirmed to be sperm negative with mass spectrometry and microscopic analysis. This data supports the use of current immunochromatographic assays for the presumptive detection of seminal fluid while also providing further support for the improved specificity of alternative serological approaches using mass spectrometry identification of biological targets. With regard to the relationship between quantitative levels of target seminal fluid peptides and the ability to generate STR profiles from vaginal swabs collected at various post coital intervals, a total of 61 post-coital samples were tested. Of these, 48 samples had a seminal fluid target greater than the limit of quantitation for the mass spectrometry assay and 26 produced an STR (n=9) and/or Y-STR (n=10) profile. A correlation between peptide quantitation and ability to generate a genetic profile was unable to be determined from this initial sample set. Overall, however, it has been demonstrated that the use of proteomic mass spectrometry for the identification of seminal fluid targets (with its enhanced sensitivity and specificity) would enable forensic practitioners to make better use of serological information during the analysis of challenging sexual assault samples.