The continuing development of efficient high-performance liquid chromatography (HPLC) protocols is critical for all areas of biomedical science and biotechnology. The four major areas under investigation in our laboratory are: (1) development of novel HPLC and capillary electrophoresis (CE) separation methods for peptides and proteins; (2) utility of reversed-phase chromatography (RPC) to monitor folding and stability of peptides and proteins; (3) to correlate peptide elution behavior in RPC and hydrophilic interaction/cationexchange chromatography (HILIC/CEC) with biological activity; and (4) development of automated two dimensional HPLC for peptides and proteins in proteomics applications. There is an ever-increasing demand for efficient analytical and preparative purification techniques for peptides and proteins. Thus, HPLC is used routinely in our laboratory for purification of native proteins and protein fragments, recombinant proteins and synthetic peptides, the latter of which are a class of compounds with increasing therapeutic importance. Novel purification protocols, such as mixed-mode HILIC/CEC with the development of volatile mobile phases, will continue to rival RPC. A generally applicable one-step RPC protocol for recombinant proteins from whole cell lysates will represent a considerable advance on existing multi-step approaches. Also, our novel sample displacement chromatography (SDC) approach, when scaled down to microbore and capillary RPC columns, will allow efficient purification of even limited amounts (0.5 mg to 10 mg) of crude synthetic peptides. Both RPC and HILIC/CEC represent potent monitors of polypeptide structural characteristics and it will be possible to correlate elution behavior with amphipathicity and hydrophilicity/hydrophobicity of antimicrobial alpha-helical and beta-sheet peptides for SAR (structure activity relationships) studies. Thus, we are showing the advantages of HPLC in the understanding of the action of peptide antimicrobial mechanisms, a vital link in the de novo design and development of novel antibiotics as a response to the increasing bacterial resistance to traditional antibiotics. The overall aim of proteomics is the systematic identification and quantification of all proteins expressed by an organism at a certain time and under certain conditions. Proteomics is a multidisciplinary research activity wherein separation science (including multidimensional HPLC and CE) and mass spectrometry will continue to play pivotal roles.