Due to the public health concerns of endocrine-disrupting chemicals, there is an increasing demand to develop improved high-throughput detection assays for enhanced exposure control and risk assessment. A substrate-free, autobioluminescent HEK293ARE/Gal4-Lux assay was developed to screen compounds for their ability to induce androgen receptor (AR)-mediated transcriptional activation. The assay was validated against a group of 40 recommended chemicals and achieved an overall 87.5% accuracy in qualitatively classifying positive and negative AR agonists. The HEK293ARE/Gal4-Lux assay was demonstrated as a suitable tool for Tier 1 AR agonist screening. By eliminating exogenous substrate, this assay provided a significant advantage over traditional reporter assays by enabling higher-throughput screening with reduced testing costs while maintaining detection accuracy.