Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. (C) 2015 Published by Elsevier B.V.