Drug-facilitated sexual assaults are a public health and safety concern. Liquid chromatography paired with tandem mass spectrometry is theoretically capable of detecting the scope of drugs commonly encountered in these types of cases. An analytical method was developed for the quantitative analysis of 40 drugs designated by Academy Standards Board 121 “Standard for the Analytical Scope and Sensitivity for Forensic Toxicological Testing of Urine in Drug Facilitated Crime” (ASB 121). The targeted analytes spanned a range of drug classes including antidepressants, antihistamines, barbiturates, benzodiazepines, cannabinoids, stimulants, and opioids. The final method utilized supported liquid extraction, followed by liquid chromatography tandem mass spectrometry with electrospray ionization in simultaneous positive and negative mode. Multiple reaction monitoring allowed quantification of analytes along with stable isotope internal standards. Validation parameters assessed included linearity, bias, precision, limit of detection, lower limit of quantitation, interference, and ion suppression or enhancement. The utilized sample preparation method was able to extract 36 of the 40 target analytes and the developed analytical method was able to detect and quantify all analytes to the sensitivities required by ASB 121.