The growth of auxotrophic bacteria remains the method of choice for the determination of biologically active folate metabolites in plasma. This report describes a microbiological assay for folates adapted to use disposable 96-well plates and an automatic plate reader. The modifications in the assay decreased reagent costs and made the analysis of hundreds of samples per day possible with a sensitivity limit of 10 fmol of (6S)-5-formyltetrahydrofolic acid. This limit compares favorably with that of previously reported, more laborious methods. The unnatural 6R diastereomer of 5-formyltetrahydrofolic acid did not interfere with the microbiological assay of the natural 6S diastereomer.