Development of efficient and reliable fermentation processes for protein pharmaceuticals is aided by the availability of accurate quantitative and qualitative product analyses. We have developed a variety of single and dual column chromatographic separations that meet the needs of process development and examples will be provided of how the resulting data has been used to optimize the culture process. For single column methods, reversed-phase chromatography has been the most versatile, permitting the reliable quantitation of many yeast, Chinese hamster ovary (CHO) cell and Escherichia coli-expressed products in the matrix of culture broth or cell extract. Analysis of secreted human growth hormone synthesized in E. coli, along with clipped and unprocessed forms, will be discussed. Another reversed-phase assay for direct analysis of a peptide product (B-chain relaxin) and its degradation products secreted into E. coli fermentation medium has allowed the purification of the responsible protease. Cation-exchange has proven extremely useful for the direct analysis of antibody fragment synthesized in E. coli, allowing the separation and quantitation of the desired Fab' and Fab'2, as well as the unwanted products of glutathione addition and translational read-through. Assay development is often complicated by the presence of host proteins with chromatographic behavior that is similar to that of the product. Commercial instrumentation now permits the facile development of multidimensional chromatographic assays. We show examples of coupled receptor affinity-reversed-phase assays for a mistranslation product and for covalent multimers of E. coli-synthesized lymphotoxin.