Prostate cancer (PCa) is the second leading cause of male cancer-related deaths in the United States. Biomarkers for effective early diagnosis and prognosis of prostate cancer are still lacking. The pre-mature forms of prostate specific antigen (PSA), proPSAs, have been shown to be associated with PCa in patient plasma or sera. Herein, we report the development of highly specific targeted mass spectrometry assays for simultaneous quantification of [-2, -4, -5, -7]proPSA isoforms in human urine. The diagnostic utility of these isoform markers was evaluated with a cohort of 30 well-established clinical urine samples. The two [-2, -4]proPSA isoforms, are highly promising in discriminating cancer from non-cancer patients with p values of 0.01 and 0.03, respectively. The other two [-5, -7]proPSA isoforms have p values of 0.07 and 0.04, respectively. Under the 95% confidence interval, the combination of [-2]proPSA and [-4]proPSA isoforms yields the area under curve (AUC) of 0.86 and the AUC value for the combined all 4 proPSA isoforms was calculated to be 0.85. Due to its highest abundance among all the proPSA isoforms in human urine as well as its being the most promising proPSA marker, we have further verified [-2]proPSA, the dominant isoform, in an independent cohort of 34 clinical urine samples with p<0.0019 for distinguishing PCa patients from healthy controls. Validation of these proPSA isoforms in large-scale cohorts as well as with increased SRM sensitivity is needed to demonstrate their potential clinical utility, including their prognostic values for predicting the aggressive forms of PCa.