To develop a semi-quantitative method for prostate cancer diagnosis and to validate this technique in clinical protocols with the use of multiplex RT-PCR assays for five different biomarkers associated with carcinogenesis, including the PCA3 gene.AR, SRD5A2, KLK2, PSMA, and PCA3 transcripts were analyzed by multiplex RT-PCR assay in 73 prostatic tissue samples from patients with prostate cancer (PCa) and benign hyperplasia (BPH).Significant differences were observed between cancerous and hypertrophic tissues in the relative expression of these genes. AR, KLK2, PSMA, and PCA3 genes displayed increased transcriptional levels in the cancer specimens; on the other hand, SRD5A2 mRNA levels were higher in the BPH samples.Our results suggest that the most promising marker for PCa diagnosis was positive PCA3 detection associated with serum PSA levels, which showed 28-fold higher chances for cancer occurrence, with 92% specificity and 94% positive predictive value.