Reversed-phase HPLC (RP-HPLC) is one of most important techniques for protein separations and the method of choice for peptide separation. RP-HPLC has been applied on the nano, micro, and analytical scale, and has also been scaled up for preparative purifications, to large industrial scale. Because of its compatibility with mass spectrometry, RP-HPLC is an indispensable tool in proteomic research. With modern instrumentation and columns, complex mixtures of peptides and proteins can be separated at attomolar levels for further analysis. In addition, preparative RP-HPLC is often used for large-scale purification of proteins. This unit provides protocols for packing and testing a column, protein separation by use of gradient or step elution, desalting of protein solutions, and separation of enzymatic digests before mass spectrometric analyses. A protocol is also provided for cleaning, regenerating, and storing reversed-phase chromatography columns.