Many candidate biomarkers for diagnosis of prostate cancer have been investigated, but prostate-specific antigen (PSA) testing remains the frontline test for both mass screening and individual clinical testing. Although the PSA test is cost-effective, analytically reliable, and flexibly high throughput, it has a very weak correlation with prostate malignancy. This has resulted in over-diagnosis and over-treatment of patients leading to costly economic, social, and psychological impacts. PSA testing lacks the ability to molecularly characterize prostate diseases and define aggressiveness and lethality, which are necessary to influence choice of treatment. Therefore, newer molecular tests are beginning to replace the PSA tests. The prostate cancer antigen 3 test has shown superiority and is now widely used. The recently reported sarcosine urine test, the already delineated TMPRSS2: ETS fusion genes, the glutathione-S-transferase P1 serum marker, and enhancer of zeste homolog 2 biomarker may also help improve diagnosis and prognostication of prostate cancer. The analytical trend is toward a multiplex testing format using molecular and/or proteomic techniques that are reliable, accurate, reproducible, and ensure rapid quantitation. Therefore, validation of these newer biomarkers and their assays are necessary for both large-scale clinical trials and clinical utility.