The cellular thermal shift assay (CETSA) provides a means of understanding the extent to which a small molecule ligand associates with a protein target of therapeutic interest, thereby inferring target engagement. Better analytical detection methods, including mass spectrometry, are being implemented to improve quantitation within these assays, providing both absolute quantitation and a very high analyte specificity. To understand the target engagement, and hence inhibition, of the protein dipeptidyl peptidase 9 (DPP9) in rat tissue, CETSA experiments, coupled with single-pot, solid-phase-enhanced sample preparation ("SP3") and absolute quantitation by high-resolution mass spectrometry, demonstrated a temperature-dependent "melting curve" by ex vivo incubation of compound with rat tissue and further demonstrated in vivo engagement by a dose-dependent response to treatment. These experiments illustrate the ability to extend the CETSA to in vivo dosed-animal samples using absolute quantitation of DPP9 by mass spectrometry and demonstrate a viable path for interrogating therapeutic molecules for drug discovery.