Modification of an enzyme assay for 5-fluorocytosine, from an end point to a rate analysis, has resulted in a method that can provide results within 10 minutes. The modification also rendered the assay less susceptible to interference from bilirubin and triglycerides. This obviated the need to correct 5-fluorocytosine results for icteric or moderately lipemic samples. The performance history of the enzyme rate method has shown that it is reliable, quick, cost-effective, and suitable for use in a clinical laboratory.