A microplate-based HPLC assay for transketolase is described for rapidly determining substrate and product concentration suitable for optimisation of biocatalytic process conditions and screening directed evolution libraries. Transketolase catalyses the enantioselective carbon-carbon bond formation of chiral keto-diol products. The assay was used to determine dissociation constants for the two cofactors required by transketolase with 5-11% error. The preparation of samples by microplate-based fermentation, cell lysis, addition of cofactor, addition of substrates was also evaluated and optimised for increased transketolase activity. The whole process enables 3-fold improved enzyme variants to be identified from a single measurement.