Typical steps involved in the development of a mass spectrometry-based targeted assay include: (1) selection of surrogate or signature peptides corresponding to the targeted protein or modification of interest; (2) iterative optimization of instrument and method parameters for optimal detection of the selected peptide; (3) method development for protein extraction from biological matrices such as tissue, whole cell lysates, or blood plasma/serum and proteolytic digestion of proteins (usually with trypsin); (4) evaluation of the assay in the intended biological matrix to determine if endogenous analyte is detected; if target analytes are of low abundance, an enrichment step may be necessary; and (5) assay validation applying fit-for-purpose principles on biological samples to characterize assay performance. The incorporation of standard operating procedures (SOPs) and method documentation are critical for maintaining the reproducibility and transferability of measurements. Bioinformatic support for such an endeavor is undoubtedly critical in assay configuration and data analysis. Publicly available software/algorithms and peptide/protein databases useful for successfully configuring targeted mass spectrometry-based assays include Skyline, PeptideAtlas, SRMAtlas, PRIDE, etc. Once developed and characterized, these assays can readily be deployed and adopted across laboratories for the precise quantification of the targeted peptide/protein.