There is intense interest in determining the absolute abundance of specific proteins in complex mixtures, for example in the area of disease biomarker discovery. We have developed a set of protein-tagging reagents called visible isotope-coded affinity tags (VICAT reagents) that contain a protein-tagging reagent for reaction with cysteine SH groups, a visible probe for monitoring the chromatographic behavior of the target peptides, a photo-releasable biotin affinity tag for selective capture and release of tagged peptides, and a heavy isotope tag for differentiating analyte from internal standards. These reagents are used together with isoelectric focusing and reverse-phase microbore chromatography/electrospray ionization/tandem mass spectrometry to determine the absolute abundance of a set of target proteins in a complex mixture, such as a cell lysate. VICAT reagents should also be useful for detecting low abundance proteins in biological fluids such as serum, and for the detection of posttranslational protein modifications and different splice variants.