Background <p>Apoplastic pH is a central regulator of plant growth, development, and environmental adaptation, influencing cell expansion, nutrient uptake, and extracellular signaling. Many studies have successfully used HPTS to monitor relative changes in apoplastic pH in plants. At the same time, research increasingly targets pH-dependent biochemical and biophysical processes. Many enzymatic activities, ion binding events, and receptor–ligand interactions depend on defined proton concentrations. Accordingly, the development of reliable approaches to measure absolute pH in living tissues is gaining importance.</p> Methods <p>A calibration-based workflow was developed to enable quantitative assessment of absolute apoplastic pH using ratiometric HPTS imaging. The approach integrates a simplified two-point normalization strategy with an in-vitro derived sigmoidal calibration model, thereby minimizing the need for extensive in-vivo calibration curves. Confocal imaging was performed using HPTS excited at two wavelengths followed by ratiometric image processing. Data analysis is supported by a custom Fiji plugin, Ratio2pH, which converts ratiometric images into pixel-resolved maps of absolute pH.</p> Results <p>In vitro characterization revealed a robust, non-linear relationship between normalized HPTS ratios and pH, enabling accurate pH estimation within the physiologically relevant range of pH 5.0–7.0. When applied in-vivo to Arabidopsis thaliana roots, the workflow yielded extracellular pH estimates consistent with the pH of the incubation medium and detected reproducible pH shifts in response to pharmacological treatments.</p> Conclusions <p>This workflow enables reproducible, spatially resolved measurement of absolute apoplastic pH in living plant tissues. By combining a simplified calibration strategy with accessible image analysis tools, it facilitates quantitative extracellular pH measurements and their integration into biochemical and biophysical analyses.</p>