The rate-limiting step concept has been widely used in anaerobic digestion processes as well as in many different biological processes. Sometimes it is described as part of the study introduction, but many times is used to support the specific objectives of the work. However, there is no clear consensus of how to define this step or how to determine it. This review presents a critical revision of several methods and procedures proposed to determine the rate-limiting step in the anaerobic digestion field. The review is structured as follows: first, the justification of this revision is presented, followed by the very basic original definition and the latest applications in determining the rate-limiting step. It ends with a discussion about the existing methodologies and the possible directions for addressing this issue. Different experimental approaches have been used for determining the rate-limiting step which, in general terms, can be grouped into two procedures: steady states and batch test evaluation. Modeling applications have rarely been employed despite them allowing a more detailed picture of the anaerobic digestion system Copyright (c) 2012 Society of Chemical Industry