How would you confirm a hit is not an assay interferent?

Why

First confirm sample identity, purity and reproducible concentration-response behaviour using independently prepared stock. Repeat the effect with a detection principle that does not share the original reporter: a fluorescent readout can be confounded by autofluorescence or quenching. Include reporter-only and no-target counter-screens.

Check colloidal aggregation with validated detergent tolerance, enzyme concentration dependence or direct particle detection, and consider reactive chemotypes. In cell assays, track viability and morphology to distinguish the desired pathway signal from generalized damage. Follow a biochemical hit with direct physical binding or cellular target engagement when feasible, and perturb the target genetically.

For example, a bright compound that increases fluorescence without target protein should be triaged as likely optical interference. No single counter-screen settles every mechanism, so interpret the combined evidence.