We report that an MDA‐kb2 human cell line that has both endogenous androgen (AR) and glucocorticoid (GR) receptors and a stably transfected MMTV‐luciferase (Luc) can be reliably used to screen for substances that may induce or inhibit gene activation via AR‐or GR‐mediated pathways. Confirmation assays are essential components of our protocols to verify that induction or inhibition of luciferase expression occurs through AR receptor activation or inactivation. A pure AR antagonist (nilutimide: NIL) is used for agonist confirmation and a pure AR agonist (R1881 or dihydrotestosterone: DHT) is used for antagonist confirmation. DHT and R1881 have EC50’s of 1.5×10‐10M and 8×10‐11M, respectively, and NIL an IC50 of 1×10‐7M. We use progesterone and cycloheximide as the negative controls in AR agonist confirmation and antagonist confirmation assays, respectively. Although progesterone is commonly reported as an AR agonist in the literature, our use of a confirmation assay shows that progesterone is a false positive that makes a good negative control. Previously published androgen antagonist assays lacking a confirmation assay have increased false positives due to cytotoxicity, protein synthesis inhibition, or other cell cycle disruption. While earlier studies have managed to control for cytotoxicity, using cycloheximide as the negative antagonist control eliminates false positives due to other factors. We tested 30 coded chemicals supplied by NICEATM using protocols that included confirmation assays, and found no false positives or negatives compared to NICEATM/ICCVAM meta‐analyses. The assay protocols using DHT as the positive control are currently undergoing a single lab validation study.