Background <p>Trametinib (TRT) is a kinase inhibitor displaying extensive erythrocyte-plasma partitioning (EPP). Plasma concentrations have been evaluated in most pharmacokinetic studies and for therapeutic drug monitoring (TDM), with controversial outcomes. We investigated EPP to evaluate its impact on TRT pharmacokinetics and laboratory assays.</p> Methods <p>TRT concentrations were monitored in spiked whole blood, in plasma and erythrocytes separated from spiked whole blood, and in plasma spiked directly. The erythrocyte-plasma partitioning coefficient (K_e/p) was established at 38&#xa0;°C, room temperature (RT), and 2–8&#xa0;°C. TRT concentrations and K_e/p were assessed in samples collected from a pediatric patient receiving TRT and in the steady state. Assays were performed using liquid chromatography-mass spectrometry and an in-house method validated for plasma TRT assays.</p> Results <p>TRT was chemically stable in whole blood and plasma at RT and 2–8&#xa0;°C. The TRT content of erythrocytes varied by concentration and storage temperature. At 20 ng/mL, K_e/p increased remarkably at RT and 38&#xa0;°C, but remained unchanged at 2–8&#xa0;°C. At 60 ng/mL, K_e/p increased throughout the 4-hour time frame, though to a considerably lesser extent. In in vivo samples, K_e/p showed a strong multivariate correlation with time after dose intake and the product of dose and hematocrit at 7.33–15.7&#xa0;h postdose (<i>r</i> = 0.987). TRT also exerted temperature-dependent diffusion from erythrocytes into TRT-free plasma.</p> Conclusions <p>EPP of TRT is related to concentration, temperature, and time in the therapeutic concentration range. Parallel whole blood and plasma TRT assays may therefore be useful for clinical pharmacokinetic studies and TDM.</p>