To establish a rapid, specific, sensitive and simple assay method for doxorubicin (DXR) in body fluid, monoclonal antibodies (MAbs) against DXR were generated by immunizing mice with keyhole limpet hemocyanin-DXR conjugate, cell fusion, and a one step, time saving screening ELISA method using aminoplate-coupled DXR via a glutaraldehyde bridge as solid phase antigen. Inhibition ELISA for DXR-immunoassay was established using anti-DXR MAb of the best producer (2E9) and aminoplate-coupled DXR as antigen and DXR ranging from 50 pg to 50 ng in the body fluid or in the cell extract could be detected. MAb 2E9 cross-reacted to various degrees to anthracycline compounds, such as some DXR analogues and derivatives, but did not recognize anthracene and anthraquinone structures.