Purpose <p>This study was designed to analyse the influence of temperature, pH and storage time on unbound fractions of PHT and VPA.</p> Methods <p>The influence of ultrafiltration (UF) temperature on measured unbound fractions of PHT and VPA in spiked samples was evaluated in a single laboratory experiment and in data from a national external quality control (EQC) database. The influence of pH adjustment with phosphate buffered saline (PBS) on measured unbound fractions of PHT and VPA was investigated in patient samples. The influence of storage time on unbound fractions of PHT and VPA was examined in patient samples by performing UF at various time points.</p> Results <p>Performing UF at a temperature of 37&#xa0;°C compared to 20&#xa0;°C significantly increased (<i>p</i> &lt; 0.05) the measured unbound fractions for both PHT (range 14.8–26.3%) and VPA (range 5.7–16.0%) in the single laboratory experiment. Consistent with these findings, reported unbound fractions in the ECQ database of PHT were significantly higher with UF at 37&#xa0;°C compared to room temperature. For VPA, this was not the case. However, for both drugs, the number of laboratories performing UF at 37&#xa0;°C was limited (<i>n</i> = 2–4). Adjustment of pH with PBS was unfeasible. After 4 days of storage at 5&#xa0;°C, the unbound fraction of PHT seemed to remain stable whereas the unbound fraction of VPA appeared to increase (27.4%) (<i>n</i> = 3).</p> Conclusion <p>Higher UF temperatures led to an increase in measured unbound fractions of both PHT and to a lesser extent VPA. Storage time of plasma samples might influence the unbound fraction of VPA.</p>