The first step in formulating a defined medium is to conduct a thorough search of the scientific literature. If a defined medium formulation is located that might be compatible with the intended cell system, a pilot study should be carried out to evaluate the general performance of the medium. Depending on the initial data obtained from this study, individual components of the medium and their concentrations may need to be manipulated (added/subtracted, increased/decreased) to obtain the desired results. Also, sometimes the basal medium or proportions of basal media must be changed. Because the formulation of a defined medium is a circular process, alteration of the basal medium type or ratio of basal media will necessitate redoing all of the previous addition/subtraction and optimization steps. Revalidation must also be done if vendors of components are changed or whenever different cells or cells of other ages are used in the system. This paper presents a brief procedure for formulating a defined media and an overview of the application of two defined media in muscle cell culture.