This protocol describes a 96-well-plate-based, enzymatic assay for reliably estimating ethanol concentrations in experimental samples in one hour. In the presence of excess NAD+, alcohol dehydrogenase (ADH) is employed to convert ethanol to acetaldehyde. The concomitant conversion of NAD+ to NADH is monitored via increased absorbance at 340 nm. When highly accurate analytical techniques (such as high performance liquid chromatography) are not necessary, or are too costly or low-throughput, this assay offers reliable, inexpensive, and rapid detection of ethanol concentrations. This assay is useful for applications such as determining relative ethanol production from microbial fermentations, and detecting ethanol evaporation from media.