Despite a ban by the European Union, the use of anabolic steroids and repartitioning agents in cattle is still occasionally observed. Due to continuing improvements in analytical techniques, very low detection limits for individual compounds have been achieved. In response to these developments, cocktails composed of several steroids have been applied, thus hampering detection due to lower levels of the individual compounds. Bioassays capable of measuring the integrated effect of cocktails might therefore provide valuable additional tools in controlling the use of illegal anabolics. We investigated the feasibility of using the AR-LUX assay to detect the presence in cattle urine of growth promoters that exert their effects via androgen response elements (AREs). The AR-LUX assay is based on a human cell line featuring a luciferase reporter gene under transcriptional control of an authenticated ARE. Several column purification and liquid/liquid extraction methods were investigated to optimize the efficiency of anabolic compounds extraction and minimize cytotoxic effects of the urine matrix. The AR-LUX assay was found to be applicable to the detection of anabolic steroids excreted in urine samples with a discriminatory power similar to that of GC-MS analysis. Finally, some liquid products probably destined for growth-promoting purposes confiscated outside the Netherlands were analyzed. Although common chemical-analytical methods did not detect any anabolic steroids in these samples, the presence of compounds activating ARE-mediated gene expression was clearly established.