The eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size-exclusion chromatography (HP-SEC), ion-exchange chromatography (HP-IEX), normal phase chromatography (HP-NPC), hydrophobic interaction chromatography (HP-HIC), reversed-phase chromatography (RP-HPLC), hydrophilic interaction chromatography (HP-HILIC), immobilized metal ion affinity chromatography (HP-IMAC), and biospecific/biomimetic affinity chromatography (HP-BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP-MMC), charge transfer chromatography (HP-CTC), or ligand-exchange chromatography (HP-LEC) are described. The unit includes overviews of the system requirements and planning strategies for set-up of the systems.