Purpose: Pemetrexed is a cytotoxic drug used for the treatment of lung cancer and mesothelioma. The use of a low test dosing of cytotoxic drugs may aid in dose individualization without causing harm. The aim of this proof-of-concept study was to assess if the pharmacokinetics (PKs) of a test dose could predict the PKs of a therapeutic pemetrexed dose. Methods: Ten patients received both a low test dose (100 μg) and a therapeutic dose of pemetrexed after which plasma concentrations pemetrexed were measured. PK analysis was performed by means of nonlinear mixed-effects modelling. The predictive performances of test dose clearance and renal function towards a therapeutic dose were assessed. Results: The PKs of a pemetrexed test dose were best described by a one-compartment model with linear elimination. A high variability in the administered dose was observed for the test dose, but not for the therapeutic dose. A statistically significant correlation between test dose clearance and therapeutic dose clearance was observed (Spearman's rho: 0.758, P = 0.02). The predictive performance of test dose clearance was worse than renal function: mean predictive error (+95% confidence interval [CI]) 53.9% (50.1-57.6%) vs 19.4% (12.4-26.4%) and normalized root-mean square error (+95% CI) 57.8% (30.5-85.1%) vs 25.7% (20.3-31.0%). Conclusion: We show that test dosing of pemetrexed is feasible, but there seems no added value for a low test dosing in the dose individualization of pemetrexed.