Many drugs are enzyme inhibitors, and enter clinical practice after a long development process. Unfortunately, the experimental quantification of enzyme activity exhibits large variance in the scientific literature. In this talk, we discuss the primary causes leading to the large variance in error estimates, for enzyme activity. We use the simplest enzyme-catalyzed reaction as a case study. The conditions under which the Michaelis–Menten equation accurately captures the steady-state kinetics of a simple enzyme-catalyzed reaction is contrasted with the conditions under which the same equation is used to estimate kinetic parameters in progress curve experiments. Our analysis shows that satisfaction of the underlying assumptions leading to the Michaelis–Menten equation are necessary, but not sufficient to guarantee accurate estimation of kinetic parameters. We derive a new condition based on time-scale separation of the linear and nonlinear portions of the progress curve that indicates when the kinetic parameters can be estimated from a single experiment, with some degree of confidence. We propose experimental conditions that allow for accurate estimation of kinetic parameters.