The in vitro characterisation of a mechanism-based enzyme inactivator (MBEI) includes determination of the maximum inactivation rate constant (k(inact)), the inactivator concentration that produces half-maximal rate of inactivation (K(I)), and the partition ratio (r). Conventional experimental protocols (CEPs) assume insignificant metabolism of the MBEI during the "pre-incubation" stage and negligible inactivation of enzyme during the "incubation" stage. The aim of this study was to evaluate the bias in the estimation of kinetic values as a consequence of these assumptions. Ranges of values of k(inact), K(I), and r for reported MBEIs were collated and data for 27 virtual compounds were generated by combining the median, high and low values of each parameter. The kinetics of the virtual compounds and of four reported MBEIs were simulated under CEP, but taking account of enzyme inactivation, metabolism of the MBEI and the probe substrate, and their interaction at relevant stages. The differences between the estimated and starting kinetic values reflect the bias introduced by the CEP in the absence of experimental error. Despite simulating a stringent experimental procedure, 19% of the estimated kinetic values of the 27 virtual MBEIs had greater than 100% bias. Simulations relating to two of the actual MBEIs indicated no bias in k(inact) and 8-33% bias in K(I). However, the bias in K(I) values of the two other compounds exceeded 98% and corresponding bias in k(inact) was greater than 300%. Thus, CEP may introduce substantial bias in estimated kinetic values for mechanism-based inhibition, and the validity of some of the reported kinetic parameters may be questionable.