Non-linear dose-exposure (supra-proportionality) occurs when plasma drug concentrations increase in a non-linear fashion with increasing dose. To predict the likelihood of this, an understanding is required of the K(M), which reflects a drug ability to saturate a specific enzyme involved in its metabolism. This study assessed the accuracy of K(M) and V(max) determinations for compounds using a substrate-depletion approach with those determined using the product-formation approach, using both recombinant human cytochrome P450 (CYP) enzymes and human liver microsomes. For the vast majority of the compounds studied, the K(M)'s using recombinant CYPs and human liver microsomes in the two approaches predicted within two-fold. Further comparisons between the K(M) and V(max)-values were made between those measured using the product-formation approach and those estimated following simultaneous fitting of the Michaelis-Menten equation to all substrate depletion plots. In each case values were comparable. In conclusion, the current study showed the substrate-depletion approach can be used to estimate K(M) and V(max) using both human liver microsomes and recombinant P450s. Estimation of these parameters during early discovery will aid in the understanding of dosages at which non-linearity may occur, but potentially aid predictions of likely clinical drug-drug interactions.