To facilitate more detailed kinetic analyses of the epoxide ring-opening reactions catalyzed by HHDHs in high throughput, we developed a quantitative pH indicator assay inspired by work from Gul and colleagues. This assay relies on the conversion of a strong acid (e.g. azide) to a weak acid (e.g. an alcoholate) during epoxide ring opening, which increases the net pH value of the reaction mixture while the reaction progresses. Using bromothymol blue (BTB, an indicator with sweeping absorption spectra giving light yellow to dark blue mixtures) and dilute MOPSbuffered reaction mixtures, we typically followed ring-opening reactions starting at around pH 7. Unlike previously described assays using pH indicators, our system returns quantitative conversion data by employing isometric normalization. Using the isosbestic point of the deprotonation of BTB, we traced the deprotonation equilibrium of the indicator back to a concentration of consumed protons via the buffer strength (see the SI for details and mathematic operations). This assay proved readily compatible with high-throughput experimentation in 96-well plates and allowed straightforward monitoring of more than 20 reactions in parallel. Although we primarily used the assay for HHDH-catalyzed epoxide ring-opening reactions in our study, we expect it to be applicable to other protonconsuming or -liberating (biocatalytic) reactions as well. A) Crystal structures of HheC (green; PDB: 1PWX) and HheG (blue; PDB: 5O30) wild type with conserved residues of sequence motifs 1 and 2 highlighted. B) Solubility data of motif 1 mutants of HheC and HheG according to SDS-PAGE analysis (Figure ). C) Activity screening data (dehalogenation and epoxide ring opening) of motif 1 mutants of HheC and HheG using qualitative pH indicator-based assays. High activity or solubility is represented by purple color, whereas no activity or insolubility is represented by yellow color. Grey color represents HheC mutants that could not be generated on genetic level.