The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However， current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious， time consuming， and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies， Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10)， respectively. PLiRT-PCR， relies on protein recognition by pairs of proximity probes (antibody-DNA conjugates) that give rise to a ligated DNA strand. The ligated DNA strand is then amplified and detected by qPCR.