Prostate cancer is the leading type of cancer diagnosed in men. Serum prostate-specific antigen levels and digital rectal exam are far from perfect when it comes to differentiation of patients with prostate cancer and benign prostatic hyperplasia. In this study, we attempt to determine whether amino acids can be used as prostate cancer biomarkers. Concentrations of derivatized amino acids and amines were quantified by liquid chromatography tandem mass spectrometry. A total of 100 urine samples from the two groups including samples provided before and after prostate massage were examined quantitatively for amino acid and amine concentrations with 50 urine samples collected from cancer patients and 50 samples from patients diagnosed with benign prostatic hyperplasia. Arginine, homoserine, and proline were more abundant in urine samples of cancer patients compared with arginine, homoserine, and proline levels determined in urine collected from patients with benign growth. We also show that sarcosine is not a definitive indicator of prostate cancer when analyzed in urine samples collected either before or after prostate massage.