© 2021 THE AUTHORS.Heterotrimeric G protein subunits Gαq and Gα11 are inhibited by two cyclic depsipeptides, FR900359 (FR) and YM-254890 (YM), both of which are being used widely to implicate Gq/11 proteins in the regulation of diverse biological processes. An emerging major research question therefore is whether the cellular effects of both inhibitors are on-target, that is, mediated via specific inhibition of Gq/11 proteins, or off-target, that is, the result of nonspecific interactions with other proteins. Here we introduce a versatile experimental strategy to discriminate between these possibilities. We developed a Gαq variant with preserved catalytic activity, but refractory to FR/ YM inhibition. A minimum of two amino acid changes were required and sufficient to achieve complete inhibitor resistance. We characterized the novel mutant in HEK293 cells depleted by CRISPR–Cas9 of endogenous Gαq and Gα11 to ensure precise control over the Gα-dependent cellular signaling route. Using a battery of cellular outcomes with known and concealed Gq contribution, we found that FR/YM specifically inhibited cellular signals after Gαq introduction via transient transfection. Conversely, both inhibitors were inert across all assays in cells expressing the drug-resistant variant. These findings eliminate the possibility that inhibition of non-Gq proteins contributes to the cellular effects of the two depsipeptides. We conclude that combined application of FR or YM along with the drug-resistant Gαq variant is a powerful in vitro strategy to discern on-target Gq against off-target non-Gq action. Consequently, it should be of high value for uncovering Gq input to complex biological processes with high accuracy and the requisite specificity.