We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances. This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay (in the form of relative fluorescence units, RFU which are then converted to microgram protein using bovine serum albumin standards) with which to normalize luciferase activity (in the form of relative light units, RLU) of cell lysates without requiring any transfer of the cell lysates. This data demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays.