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Prostate Cancer (PCa) diagnosis is currently hampered by the high false-positive rate of PSA evaluations, which consequently may lead to overtreatment. Non-invasive methods with increased specificity and sensitivity are needed to improve diagnosis of significant PCa. We developed and technically validated four individual immunoassays for cathepsin D (CTSD), intercellular adhesion molecule 1 (ICAM1), olfactomedin 4 (OLFM4), and thrombospondin 1 (THBS1). These glycoproteins, previously identified by mass spectrometry using a Pten mouse model, were measured in clinical serum samples for testing the capability of discriminating PCa positive and negative samples. The development yielded 4 individual immunoassays with inter and intra-variability (CV) <15% and linearity on dilution of the analytes. In serum, ex vivo protein stability (<15% loss of analyte) was achieved for a duration of at least 24 hours at room temperature and 2 days at 4°C. The measurement of 359 serum samples from PCa positive (n = 167) and negative (n = 192) patients with elevated PSA (2-10 ng/ml) revealed a significantly improved accuracy (P <0.001) when two of the glycoproteins (CTSD and THBS1) were combined with %fPSA and age (AUC = 0.8109; P <0.0001; 95% CI = 0.7673-0.8545). Conclusively, the use of CTSD and THBS1 together with commonly used parameters for PCa diagnosis such as %fPSA and age has the potential to improve the diagnosis of PCa.