As a steroidal drug intermediate, 16,17-epoxysterol plays an important role in pharmaceutical steroid synthesis. Current methods for epoxysterol determination normally require expensive equipment, are highly labor intensive, and are not suitable for a high-throughput system. Here we report an improved colorimetric assay with the potential to detect 16,17-epoxysterol at high throughput. The assay was developed by a colorimetric reaction using picric acid and validated in a 96-well microtiter plate format, which represents a simple and reliable assay for 16,17-epoxysterol. This method has a high potential application for the discovery of steroidal drugs.