Metabolic flux analysis with its ability to quantify cellular metabolism is an attractive tool for accelerating cell line selection, medium optimization, and other bioprocess development activities. In the stoichiometric flux estimation approach, unknown fluxes are determined using intracellular metabolite mass balance expressions and measured extracellular rates. The simplicity of the stoichiometric approach extends its application to most cell culture systems, and the steps involved in metabolic flux estimation by the stoichiometric method are presented in detail in this chapter. Specifically, overdetermined systems are analyzed since the extra measurements can be used to check for gross measurement errors and system consistency. Cell-specific rates comprise the input data for flux estimation, and the logistic modeling approach is described for robust-specific rate estimation in batch and fed-batch systems. A simplified network of mammalian cell metabolism is used to illustrate the flux estimation procedure, and the steps leading up the consistency index determination are presented. If gross measurement errors are detected, a technique for determining the source of gross measurement error is also described. A computer program that performs most of the calculation described in this chapter is presented, and references to flux estimation software are provided. The procedure presented in this chapter should enable rapid metabolic flux estimation in any mammalian cell bioreaction network by the stoichiometric approach.