While proteasome inhibition is a validated therapeutic approach for multiple myeloma ( MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (Pro CISE) assay to quantify proteasome subunit occupancy in samples from five phase I/ II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15-56 mg/m2), dose-dependent inhibition of c20S and i20S chymotrypsin-like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells ( PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow-derived CD138+ tumour cells. Carfilzomib-induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near-complete recovery between cycles. Importantly, the Pro CISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response ( n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. Pro CISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes. [ABSTRACT FROM AUTHOR]