In the field of forensic biology, the term “time since intercourse (TSI)” is used to describe the approximate time elapsed between an alleged sexual assault and the collection of a Sexual Assault Evidence Collection Kit (SAECK) from a victim. The estimation of TSI, or Post Coital Interval (PCI), can be crucial information for particular cases in which the time between offense and the collection of a SAECK is in question. Oftentimes, forensic scientists must evaluate the significance of biological test results from evidence in SAECKs, but the variability in current literature complicates interpreting these results. Developing a reliable framework to estimate TSI based on a more extensive review of forensic casework would provide investigators with a fundamental tool for estimating a general timeline in which the offense occurred. This information may play an important role in supporting or refuting a narrative, or weighing the significance of the evidence at hand. This study aims to develop a dependable framework for estimating TSI in living victims based on casework received by Boston Police Department (BPD) Crime Laboratory, Boston, MA. Additionally, this study seeks to determine if any significance exists between the victim’s reported post coital activities and the collection of evidence, including the presence of intact sperm cells. The need to expand research on estimating TSI for sexual assault victims using actual forensic casework is crucial to provide a more reliable method for TSI estimation, compared to previous studies, which have generally been based on fertility studies. Between the years of 2009 and 2017, over 1,800 reported SAECKs were submitted to the Boston Police Department for evidence processing. More than 500 of these kits met the qualifications for this study, including: a living victim, smear slides prepared by a medical professional, and the identification of sperm cells during original kit processing. In order to estimate TSI, the smear slides from these cases were microscopically examined for the presence of intact sperm cells with the aid of Kernechtrot Picroindigocarmine (KPIC) stain. Based on casework received by the BPD, the maximum TSI reported for observing intact spermatozoa on vaginal smear slides was 105 hours, with an average collection time of 15 hours. The maximum TSI in which intact spermatozoa were observed on anorectal smear slides was 17.75 hours, with an average collection time of 7.9 hours. The average collection time in which intact spermatozoa on oral smear slides were observed was 6.9 hours, with a maximum reported TSI of 13.5 hours. Moreover, data from this study indicates a positive relationship between the total number of post coital activities completed before kit collection and the passage of time. Overall, this study provides reliable evidence based on actual casework samples for more accurately estimating the timeframe in which sperm evidence can be recovered after intercourse in living victims of sexual assault crimes.