Multiple approaches for screening large sets of compounds for a specific function are of growing interest. The use of substrate mixtures to characterize the specificity of enzymes has been limited so far to compounds with similar kinetic parameters, because the data were analyzed by applying the kinetics of two competing substrates. In this study we introduce a statistical method for the analysis of reactions with many competing substrates which makes use of the specific features of multiple substrate kinetics. It is assumed that the relative concentrations of all substrates in a mixture can be monitored by high-performance liquid chromatography or a similar technique. Relative second-order rate constants, i.e., kcat/KM values, can be calculated for all substrates in the mixture from the resulting data set. The calculation uses the fact that there is a relationship between the concentrations of all pairs of substrates in the mixture. As a result, the precision of the calculated parameters is increased and the range of kinetic constants that can be obtained from one experiment is considerably expanded. Simulations demonstrate that the precision in the kinetic parameters increases with the number of substrates in the mixture. In fact, estimation of ratios of rate constants can be improved (or made possible) for substrates with order of magnitude differences in reactivity by adding “dummy” substrates with intermediate reactivities, even though the rate constants for dummy substrates are themselves of no intrinsic interest.