We began our study by developing a high-throughput method to facilitate the characterization of G3PSs, given that the substrates and products of G3PS-catalyzed transformations are only poorly tractable by common chromatographic techniques and existing methods (e.g. relying on radiolabelled substrates) were not sufficiently compatible with parallelized experimentation (also see the SI for a detailed review of previous analytical approaches). Since AfG3PS operates without discernible undesired hydrolysis of its substrates (Fig. ), we envisioned that tracking the release of the byproduct inorganic pyrophosphate would provide a reliable measure of reaction progress. Building on our recently disclosed module for phosphate detection by UV-spectroscopic monitoring of bromouridine phosphorolysis (PUB), we optimized an extended variant of the method (EPUB, Fig. ) for applications with G3PSs (see the SI and Fig. for details). EPUB translates the release of inorganic pyrophosphate into an observable UV signal via hydrolysis and subsequent conversion of the nucleoside X into the corresponding nucleobase Y which exhibits a redshifted absorption spectrum (Fig ). Under optimized conditions, EPUB generates continuous high-quality kinetic data on G3PScatalyzed transformations (Figs. 1d and S6-S11) and is readily compatible with high-throughput experimentation over various time scales and temperatures (Figs. and). Over the course of the work presented herein, we used EPUB for more than 2,000 individual kinetic reactions, generating over 80,000 datapoints at an assay material cost of <0.4 ct per datapoint (Table ). Although we primarily used EPUB for high-throughput kinetic experiments, we anticipate that the method is equally well suited for screening purposes in protein engineering campaigns.