The rs17632542 SNP results in lower serum PSA levels which may further mitigate against its clinical utility as a prostate cancer biomarker. Post-DRE urine is a minimally invasive fluid that is currently utilized in prostate cancer diagnosis. We have used targeted MS to detect and quantitate the variant protein in urine. Experimental Design Fifty-three urines collected after digital rectal exam (post-DRE urine) from rs17632542 genotyped individuals were processed and analyzed by LC-MS in a double-blinded randomized study. The ability to distinguish between homozygous wild-type, heterozygous, or homozygous variant was examined prior to unblinding. Results Stable-isotope labeled peptides were used in the detection and quantitation of peptides of interest in each sample. Three peptides were monitored by LC-MS using a PRM method. Analysis of the raw data using Skyline allowed for peak detection and area extraction. Using these data, groupings were predicted using hierarchical clustering in R. Accuracy of the predictions showed 100% concordance across the 53 samples, including individuals homozygous and heterozygous for the SNP. Conclusions and clinical relevance The study demonstrates that MS based peptide variant quantitation in urine could be useful in determining patient genotype with high accuracy to complement currently used PSA screening.