PARP7 is an enzyme that catalyzes mono-ADP-ribosylation (MARylation), is a critical regulator of type I interferon signaling, and has emerged as an immune-oncology drug candidate. PARP7 is a labile protein that is regulated in a proteasome-dependent manner. Indeed, endogenous PARP7 levels are undetectable by western blot in most cells. Intriguingly, treatment of cells with orthosteric small molecule inhibitors of PARP7 can increase endogenous PARP7 protein to detectable levels. This characteristic of PARP7 inhibitors could potentially be exploited to assess target engagement—and thus cellular efficacy—of PARP7 inhibitors; however, no method exists to quantitatively monitor endogenous PARP7 levels in a high-throughput manner. In this protocol, we describe an assay using a split Nanoluciferase (NanoLuc) system for quantifying endogenous PARP7 protein levels and PARP7 inhibitor target engagement in cells in a 96-well plate format. We show that this assay can be used to quantify PARP7 protein levels under various cellular treatments and can assess cellular PARP7 inhibitor target engagement. We envision this split NanoLuc PARP7 assay can be used not only for evaluating the cellular efficacy of PARP7 inhibitors in a high-throughput manner but also for uncovering the mechanisms regulating PARP7 protein levels in cells.