P450 aromatase (P450arom) is a well known target by anti-cancer drugs and toxic chemicals and efficient and convenient analytical tools are desired for. We established a convenient assay for P450arom inhibition based on an enzyme-linked immunosorbent assay (ELISA). The first step of the assay consists of a P450arom reaction, which converts a testosterone to a 17beta-estradiol using a recombinant human P450arom and a NADPH regenerating system. The second step of the assay consists of an ELISA system using a highly specific and sensitive anti-estradiol monoclonal antibody in conjunction with estradiol-3-CMO-horseradish peroxidase (E2-3-CMO-HRP). This system has advantages over other P450arom assays because it does not use radioactive ligands and because it is not subject to interference from self-fluorescing test compounds. We could successfully estimate some types of P450arom inhibitors reported before. This assay should be very useful for high throughput screening of drug candidates and endocrine disrupting chemicals via P450arom.