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The cultivation of mammalian cells in vitro requires special environmental conditions. Except primary cells taken directly from an organ, cultured cells usually derive from immortal or immortalized cell lines. The cells may be genetically engineered or manipulated by transfection or fusion techniques. The behavior of this new cell depends very much on the origin of the basal cell line. While some requirements are general for every cell, others can be very special for one cell line. All cells need isotonic conditions of about 300 mosmol/kg. Usually the cells need oxygen and also a carbon source (mostly glucose). As the cells cannot synthesize the essential amino acids and vitamins, they have to be supplied. Some additional components may basically not be needed for surveillance, but they can support the cells to optimize cell growth and productivity. This knowledge leads to complex media formulations that seldom contain less than 40 different components. During optimization of culture conditions and process strategies a detailed analysis of the nutrient requirements has to be performed. Many different analysis methods have to be established in a laboratory to comply with this extensive task. In this article methods for almost all media compounds are described. All methodsthat are described in detail in this article, were developed, adapted, or checked in the laboratory for suitability of analysis of cell culture media and supernatants. They should be easily transferable to similar laboratory conditions. In this article, the methods for the analysis of proteins are only very briefly described. For each protein, special conditions and methods may apply. In general, the characterization of proteins includes a variety of steps. Methods for the analysis of amino acid sequence, glycosylation, molecular weight, heterogeneity, and activity have to be employed. A description of all these procedures would be beyond the scope of this article. For this reason, protein separation methods such as electrophoresis and protein and peptide chromatography are not included here. For almost all suppliers of special equipment or chemicals cited in this article, a listing of the home pages (World Wide Web) can be found at the end of this article. All other products may be delivered by local suppliers. When a method is described in detail, an equivalent apparatus of any supplier may also be suitable. Keywords: off-line analysis; cell culture; methods; glucose; lactate; ammonia; amino acids; vitamins; inorganic ions; fatty acids; cholesterol; pyruvate; proteins