Targeted proteomics assays such as those measuring end points in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included three biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents, etc.) and a ~50-fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of nine compounds.