The absolute quantification of proteins involved in drug metabolism is a new area that is gaining in popularity and utility. By far the most often used strategy is the surrogate peptide approach. These methods rely on detection of peptides created from the protein of interest by enzymatic or chemical digestion. Typically, the protein of interest and a stable isotopically labeled internal standard peptide are digested in a relevant sample such as a microsomal fraction or plasma containing often large amounts of other proteins. The final digested sample may contain thousands of peptides derived from any combination of the proteins present in the sample matrix. The complexity of the digested sample makes selective detection a challenge, even by LC-MS/MS. Selected reaction monitoring (SRM) of HPLC separated samples is most often considered the gold standard for selectivity, however, SRM often does not have the selectivity needed to allow absolute quantification of surrogate peptides from biological matrices. Vastly improved selectivity can be realized using MS3. MS3 is a term used to describe multiple steps of mass dependent isolation and fragmentation followed by mass analysis and detection. The extra step of isolation and fragmentation associated with MS3 provides the enhanced selectivity of the technique. In this presentation, we discuss the implementation of MS3 with 40ms cycle time on the 5500QTRAP and we compare the quantitative results for six UGT isoforms measured from human liver microsomes using selected reaction monitoring and MS3. The work was done using the ABSciex QTRAP 5500 mass spectrometer and an Eksigent HT Express HPLC with 1.0 mm ID x 30 mm C18 reverse phase column and gradient elution. The enhanced selectivity of MS3 improves assay precision as measured by %CV and the assay LLOQ as compared to SRM. The improvement in signal to noise also makes automated data processing of the MS3 data for quantitative analysis of protein practical. The time required to integrate the MS3 mass chromatograms and provide quantitative results is approximately 30% of the time required to process the SRM data for the same samples.