hit confirmation
A primary screen gives you a list of suspects, not a list of culprits. Hit confirmation is the detective work that separates the two: re-testing apparent hits under stricter conditions to make sure their activity is genuine, reproducible, and caused by the compound actually engaging the target — not by chance, error, or artifact.
Confirmation typically layers several checks. The hit is re-tested in fresh material to confirm reproducibility; a full concentration series is run to confirm a proper dose-response (a real hit gets stronger with more compound, an artifact often doesn't); the compound's identity and purity are verified, since the activity could come from an impurity; and orthogonal assays using a different detection method confirm the effect isn't an artifact of the original readout.
Counter-screens and biophysical methods further check that the compound binds the target directly and selectively rather than acting through aggregation, reactivity, or off-target effects. Only compounds surviving this gauntlet earn the name 'confirmed hit' and the investment of medicinal chemistry. Skipping or rushing confirmation is how programs waste months chasing molecules that were never real.
Of 1,200 primary hits, only 140 reproduce, show a clean IC50, pass purity QC, and confirm in an orthogonal assay; these become the confirmed hits handed to chemistry.
Confirmation filters a noisy primary list down to a trustworthy set of real hits.
A defining test of a real hit is a clean concentration–response curve from freshly weighed, purity-checked material. No dose-response, or activity that vanishes on resupply, is the signature of an artifact.