Background: IsoPSA is a structure-based serum assay exploring the spectrum of possible prostate-specific antigen (PSA) isoforms. It was shown to outperform total and percent-free PSA in detecting clinically significant prostate cancer (csPCa) (grade group (GG) ≥2 on biopsy). IsoPSA reduced unnecessary biopsies and magnetic resonance imaging (MRI). We sought to compare the outcomes of eventual biopsy and imaging of surveilled patients with an initially normal or high IsoPSA, thus assessing IsoPSA’s prospective predictive ability of csPCa. Methods: We performed a single-center retrospective review of patients who underwent IsoPSA testing from 2017-present. Data was dichotomized into patients with normal (≤6) and high IsoPSA (>6). We collected the outcomes of any consequent IsoPSA and PSA test, prostate biopsy and MRI. We calculated the statistical IsoPSA’s characteristics for the prediction of csPCa on biopsy. Results: The median follow-up time of 811 patients who underwent IsoPSA testing was 18 months (IQR, 16.5-20). Among 443 patients with initial low IsoPSA, 5 (1.1%) had a csPCa on a subsequent biopsy, 19 (4.3%) subsequent high IsoPSA, 122 (27.5%) rising PSA, and 22 (5%) csPCa on MRI. Among 368 patients with initial high IsoPSA, 105 (28.5%) had a csPCa on a subsequent biopsy and 106 (28.8%) on an MRI. The sensitivity of IsoPSA to predict csPCa was 95.5%, and the NPV was 94.8%. Among 124 patients with high IsoPSA and initial negative biopsy, 110 had a subsequent negative and 14 a positive biopsy (10GG1 (8.1%), 4 ≥GG2 (3.2%)), with a respective median IsoPSA of 7.2 and 9.6 (p=0.007). An IsoPSA>10 generated an OR of csPCa of 7.2 (95%CI 2.1-25.2, p=0.0005). Conclusions: In 18-month follow-up, 1.1% of patients with normal IsoPSA developed csPCa compared to 28.5% of patients with high IsoPSA. In the cohort of patients with high IsoPSA and initially negative biopsy, 3.2% eventually developed csPCa, however, having a significantly higher IsoPSA than those who remained negative. The odds of having csPCa were 7 times higher with IsoPSA>10.