Deubiquitinases (DUBs) are important regulators of cell physiology and are emerging drug targets. However, the exact function and regulation of the majority of human DUBs remains largely enigmatic. DUB activity-based probes (ABPs) are engineered ubiquitin variants conjugated to a cysteine-reactive chemical group. We envisioned that DUB ABPs, coupled with mass spectrometry, could provide a highly sensitive and unbiased means to aid our understanding of DUB function and thus identify routes of therapeutic intervention in pathogenic processes controlled by DUBs. Recent developments in mass spectrometry and chemistry now allow for powerful multiplexing and quantitation of proteins in single experimental runs. However, current protocols are still limited by the number of samples that can be quantitatively compared with efficient throughput. Here we describe the development of an analysis platform that combines DUB ABPs with chemical multiplexing, targeted mass spectrometry, novel internal reaction standards, and a customized statistical analysis program. Our strategy allows us to quantitate changes in DUB activity across a theoretically unlimited number of samples in a high-throughput manner. We illustrate the efficacy of this technology by evaluating the activity of disease-relevant DUBs, in analyzing DUB inhibitor selectivity, and in evaluating how compounds that target other components of the ubiquitin/proteasome system impact DUB activity.