This study investigated several key parameters of deuterium exchange measurements in a dynamic vapour sorption apparatus to optimise the measurement protocol for hydroxyl (OH) group accessibility determination. The impact of changing the sample mass, the deuterium oxide (D2O) vapour exposure time and the rate of change in moisture content (dm dt−1) during the drying steps on the measured OH group accessibility were analysed. A sample mass of more than 10 mg, an exposure to D2O vapour of at least 10 h and a dm dt−1 of 0.0005% min−1 over a 10-min period during the drying steps gave the most reliable results. We also investigated the necessity of adding a method stage that eliminates the effect of inclusion compounds (ICs). The addition of an initial drying and wetting stage enabled the release of entrapped solvents.