Prostate, an overlooked target in in vitro alternative methods, is critical for male fertility. Within the EU project ReProTect, the LNCaP cell line was used as a model system to screen chemicals affecting prostate by a tiered approach integrating two toxicological endpoints: cell viability and PSA secretion. A ReProTect training set of (anti) androgenic chemicals affecting reproductive tissues were used. Androgens, and unexpectedly glufosinate ammonium, markedly increased PSA, whereas anti-androgens also increased PSA, but at a much lower magnitude than androgens. Our tiered approach properly discriminated androgenic compounds as well as yielded no false positives, as based on available toxicological evidences. The PSA secretion assay is directly linked to the prostate physiological function and it may integrate the information provided by mechanistic-based assays (i.e. AR binding and gene expression).