Recently, the number of mass spectrometry-based quantification assays has been increased, partially due to the global efforts of chromosome-centric human proteome project (C-HPP). Our goal at the Chromosome 19 Consortium is to provide novel selected reaction monitoring (SRM) assays of proteins coded on chromosome 19. We have selected the two most useful signature peptides (NSQVWLGR and HNLFEPEDTGQR) of human kallikrein-2 (hK2 - NX_P20151) and developed an SRM assay. Details of the analytical parameters, including multiple transitions by peptides, are presented. The endogenous levels of hK2 were determined in clinical samples (n = 35). The limit of quantification was also estimated by spiking heavy isotope-labeled peptides into seminal plasma samples at various concentrations (LOQ ≈ 29 ng/mL).