Compounds that exhibit assay interference or undesirable mechanisms of bioactivity (‘‘nuisance compounds’’)
are routinely encountered in cellular assays, including phenotypic and high-content screening assays.
Much is known regarding compound-dependent assay interferences in cell-free assays. However,
despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably
less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle
to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer
number of targets, but rather nuisance compounds, due to their ability to waste significant resources and
erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences
regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact
of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological