Updated project metadata. Human tissue kallikreins (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished only by enzyme-linked immunosorbent assays (ELISA). In this work, we developed a multiplex selected reaction monitoring (SRM) assay for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and then multiplex in a single assay. Assay performance was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in seminal plasma, sweat and cervico-vaginal fluid, with limits of detection ranging from 5 to 500 ng/mL. Analytical performance of the SRM assay was evaluated by measuring endogenous KLKs in relevant clinical samples and results were compared to selected ELISAs. The multiplex SRM assay was proven to be an accurate, reproducible, sensitive and highly specific alternative to the existing antibody-based assays. Finally, we used immunoenrichment-SRM and ELISA to unambiguously detect and quantify seminal plasma levels of kallikrein-4, a highly prostate-specific protein and a potential biomarker of prostate-related diseases. The presented multiplex SRM assay is an alternative analytical tool to study the biological and pathological roles of human KLKs.