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An enzyme assay measures the catalytic activity of an enzyme by following consumption of a substrate or appearance of a reaction product under specified conditions. The signal may be chemical concentration directly or a calibrated optical readout, such as absorbance or fluorescence.

For example, if product concentration increases from 0 to 6 micromolar in the first two minutes, a linear initial interval gives a rate of 3 micromolar per minute. A no-enzyme control tests whether the signal also changes without the catalyst. Comparing rates requires the same temperature, pH, substrate concentration and detection conditions; raw brightness alone is not an activity unit.

The initial interval matters because later measurements can be affected by substrate depletion, product accumulation or loss of enzyme activity. Assay-development guidance therefore recommends establishing the linear initial-rate region before comparing inhibitors or enzyme preparations.^[ref]

Harold B. Brooks coauthored practical guidance for designing enzyme assays used to test many candidate compounds. His 2012 chapter explains how enzyme kinetics, the choice of reaction substrate and controls determine whether an assay can identify reversible enzyme inhibition. Its emphasis on initial rates connects an instrument's signal to a defined biochemical reaction rather than treating an endpoint measurement as self-explanatory.^[ref]

For example, imagine an enzyme assay that starts with $100$ micromolar reaction substrate. Measuring after only $5$ micromolar has been consumed leaves its concentration approximately unchanged; measuring after $80$ micromolar has been consumed does not. The chapter recommends establishing an early linear interval with little substrate depletion, checking several enzyme concentrations and measuring background signals with appropriate missing-component controls. A later plateau can reflect exhausted reaction substrate rather than loss of enzyme activity. This is why an enzyme inhibition initial-rate assay needs a time course before it can turn a smaller signal into evidence about inhibition.^[ref]