A set of compounds (n = 30), including traditional cytochrome P450 substrates and compounds from AstraZeneca's compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CL(int), V(max), and K(m)). A depletion method previously shown to provide reliable results, the multiple depletion curves method (MDCM), was used as reference. Experiments were conducted with human liver microsomes, and samples were analyzed using liquid chromatography-tandem mass spectrometry. CL(int) estimated with the ODA were in >90% of the cases within a 2-fold difference compared with MDCM estimates. In addition, good agreement was generally seen for V(max) and K(m) estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in V(max) and K(m) estimates were generally higher than for CL(int) estimates. In addition, decreased substrate turnover considerably increased the variability in V(max) and K(m) estimates, whereas only a modest increase was observed for CL(int) estimates. The experimental design of using multiple starting concentrations for the estimation of enzyme kinetics was shown to be appropriate even when there was a limitation to the number of samples. The method allowed for good estimates of CL(int) and also for V(max) and K(m) in many cases. Hence, this approach is a good alternative for the estimation of enzyme kinetic parameters, especially if enzyme saturation and an assessment of a potential risk for nonlinear metabolism are of interest.