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[Display omitted] • Identification and absolute quantification of naturally derived prostate-specific antigen (PSA). • First absolute quantification of PSA that uses amino acid hydrolysis coupled with isotope-dilution mass spectrometry (IDMS), and enzymatic hydrolysis-IDMS simultaneously. • Support the development of high-order reference materials of PSA to ensure the accuracy, comparability, and traceability of testing in medical laboratories. Prostate-specific antigen (PSA) is a diagnostic marker for prostate cancer; however, because it is a macromolecular glycoprotein with complex and diverse isoforms, it is difficult to standardize clinical PSA detection results. To overcome this limitation, herein, naturally extracted PSA was characterized as free PSA (fPSA), and the PSA solution was successfully quantified by amino acid analysis coupled with isotope-dilution mass spectrometry (AAA-IDMS) and enzymatic hydrolysis-IDMS; the results could be traced to the International System of Units (SI) through absolutely quantified amino acids and peptides. After protein hydrolysis or digestion condition optimization, amino acids and signature peptides were detected by liquid chromatography-mass spectrometry with the multiple reaction monitoring mode. The mass concentrations of PSA obtained through AAA-IDMS and enzymatic hydrolysis-IDMS were (75.3 ± 1.5) µg/g (k = 2) and (74.7 ± 1.7) µg/g (k = 2), respectively. The PSA weighted average mass concentration was (75.0 ± 1.6) µg/g (k = 2). The consistency assessment between the two methods was successfully validated, ensuring absolute quantitative accuracy. This study lays the foundation for the development of high-order reference materials for the clinical detection of PSA, which can improve the accuracy, reliability, and consistency of clinical PSA test results. [ABSTRACT FROM AUTHOR]