Glutathione S-transferases (GSTs) form a widespread enzyme superfamily mainly involved in phase II detoxification. Differential expression of the various GST isoforms, differing in catalytic and structural properties, correlates with physiological and pathological states. Fast and simple determination of the GST profile is expected to be an important diagnostic tool in disease analysis. Here we propose a combined approach of high resolution separation techniques and electrospray mass spectrometric analyses for characterizing the spectrum of GSTs in male mouse liver. In this approach, the sensitivity and speed required for tissue GST profiling studies is achieved by tracking the reconstructed ion current of selected reporter peptides following chromatographic separation. This simple procedure, in which an affinity protein bait is followed by a chemical fragmentation and mass spectrometric analysis, could be sufficiently sensitive to detect the qualitative differences between physiological and pathological states.