Due to the complex nature of the proteome, instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation and detection becomes urgent for the identification of peptides and proteins. Newly developed techniques and equipment for separation and detection, such as nano-HPLC and multidimensional HPLC for protein and peptide separation, enabled proteomics to experience dynamic growth during the past few years. In any proteomic analysis the most important and sometimes most difficult task is the separation of the complex mixture of proteins or peptides. This review describes some aspects and limitations of HPLC, both multidimensional and one-dimensional, in proteomics research without attempting to discuss all available HPLC methods, which would need far more space than available here.