The aim of the present study was to demonstrate the applicability of a yeast androgen and estrogen bioassay in the detection of steroid esters in hair samples of animals treated with a hormone ester cocktail. The outcome of the activity screenings was critically compared with the results previously obtained with LC-MS/MS analysis. Hair samples of one pour-on treated animal, 10 ml DMSO containing 25 mg estradiol benzoate (EB), 60 mg testosterone decanoate (TD) and 60 mg testosterone cypionate (TC), were selected and analyzed with the androgen and estrogen yeast bioassay. Results showed that by the introduction of a hydrolysis step, bioassays can be used to screen for the presence of hormone esters in hair samples. Based on the difference in fluorescence responses between the non-hydrolyzed and the hydrolyzed hair samples, it was possible to detect the presence of EB up to at least 56 days after a single pour-on treatment and to detect the presence of TC and TD up to at least 14 days after the treatment. Although the LC-MS/MS analysis could detect TC and TD up to 49 days after treatment, bioassays have the advantage that they can also detect any (un)known steroid ester.