Passive proton translocation across membranes through proton channels is generally measured with assays that allow a qualitative detection of the H-transfer. However, if a quantitative and time-resolved analysis is required, new methods have to be developed. Here, we report on the quantification of pH changes induced by the voltage-dependent proton channel Hv1 using the commercially available pH-sensitive fluorophore Oregon Green 488-DHPE (OG488-DHPE). We successfully expressed and isolated Hv1 from Escherichia coli and reconstituted the protein in large unilamellar vesicles. Reconstitution was verified by surface enhanced infrared absorption (SEIRA) spectroscopy and proton activity was measured by a standard 9-amino-6-chloro-2-methoxyacridine assay. The quantitative OG488-DHPE assay demonstrated that the proton translocation rate of reconstituted Hv1 is much smaller than those reported in cellular systems. The OG488-DHPE assay further enabled us to quantify the K-value of the Hv1-inhibitor 2-guanidinobenzimidazole, which matches well with that found in cellular experiments. Our results clearly demonstrate the applicability of the developed in vitro assay to measure proton translocation in a quantitative fashion; the assay allows to screen for new inhibitors and to determine their characteristic parameters. Graphical abstract ᅟ.