Potency does not always correlate with single-dose activity. Each line corresponds with the behavior of a different molecule in the assay at different doses. HTS screens are usually performed at a single dose, shown as the black, dashed line, and the response at this dose is used to rank molecules in the primary screen for follow up (y-axis). High potency molecules reach 50% maximal activity (EC₅₀) at lower doses (x-axis), but potency can only be measured by testing the molecule at several doses. As in this example, activity at a single dose does not always correlate with potency, and activity in the primary screen does not always correlate with the potency measured in dose–response follow-up experiments.