Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine biosynthesis pathway, and a potential new antibacterial drug target. No target-based high-throughput screening (HTS) assay for this enzyme has been reported to date. Here, we optimized two colorimetric-based enzymatic assays that detect the ureido moiety of the DHOase substrate, carbamyl-aspartate (Ca-asp). Each assay was developed in a 40 μL assay volume using 384-well plates with a different color mix, diacetylmonoxime (DAMO)/thiosemicarbazide (TSC) or DAMO/antipyrine. The sensitivity and color interference of both color mixes were compared in the presence of common HTS buffer additives includingdimethyl sulfoxide (DMSO), reducing agents, detergents, and bovine serum albumin (BSA). DAMO-TSC (z'-factors 0.7-0.8) was determined to be superior to DAMO-antipyrine (z'-factors 0.5-0.6) with significantly less variability within replicates. A HTS pilot screening with 29,552 compounds from four structurally diverse libraries confirmed the quality of our newly optimized colorimetric assay with DAMO-TSC. This robust method has no heating requirement which was the main obstacle of applying previous assays to HTS. More importantly, this well-optimized HTS assay for DHOase, the first of its kind, should make it possible to screen large scale compound libraries to develop new inhibitors against any enzymes that produce ureido functional groups.