The order of the addition of substrates to and release of products from the enzyme active site, along with the establishment of relative rates of various events, defines the kinetic mechanism. These mechanisms fall into two broad groups: those where the full complement of substrates must assemble on the enzyme active site before the reaction occurs are termed “sequential mechanisms.” In the other category, a product(s) is released between additions of two substrates and are called “ping-pong mechanisms.” In this category, substitution on the enzyme active site groups occurs and hence is also known as the double displacement mechanism. The study of these mechanisms is best approached by rigorous experimental design where data are collected by systematically varying one parameter at a time. This method of reduction is in full display in enzyme kinetics. In fact, elucidating enzyme kinetic mechanisms offers the best example of how scientific hypotheses are tested. Elements of the scientific method in sequence include problem recognition → collation of available information → hypothesis building → experimentation → reasoning and deduction → refining the hypothesis. These steps are iterated to arrive at an enzyme mechanism. Finally, when experiments and measurements agree with the theory, truth is secured. In practical enzyme kinetics, this exercise translates into the following steps (Table 17.1) for the elucidation of mechanisms.