During the last ten to fifteen years a huge development in the field of analytical techniques used for deciphering the complex nature of the proteome took place. Instrumentation and methods development for sample cleanup, fractionation, preconcentration, chromatographic separation, and detection become affordable to a broad range of laboratories and new, exciting methods for both separation and detection were published and applied. Newly developed techniques and equipment for separation and detection, such as high pressure nano-HPLC and new techniques for multidimensional HPLC separation, enabled proteomics to experience dynamic growth and enter new paths. Furthermore, proteomics starts entering the “real life” and it is increasingly applied for clinical diagnostics and follow-up of patient's status during the treatment.For any proteomic analysis, one of the most important and sometimes the most difficult task is the separation of the complex mixtures of proteins or peptides prior to their detection and data analysis. 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, their benefits and shortcomings which would need far more space than available here.