Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J = 0.00) to highly promiscuous (cruzain, J = 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J = 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.