A computer program, called Pro Digest-LC, has been developed which assists scientists in devising methods of size-exclusion, cation-exchange and reversed-phase high-performance liquid chromatography for the analytical separation and purification of biologically active peptides and peptide fragments from enzymatic and chemical digests of proteins. Pro Digest-LC accurately predicts the retention behaviour of peptides of known composition, containing 2–50 amino acid residues, and simulates the elution profiles in all three modes of chromatography. In addition, Pro Digest-LC is a user-friendly program, designed as a teaching aid for both students and researchers in selecting the correct conditions for chromatography, that is, the mode of chromatography, column selection, mobile-phase selection, and has the ability to examine the effects of flow-rate, gradient-rate, and sample size on the separation. We have designed a set of peptide standards for each mode of chromatography to aid the researcher in eliminating non-specific interactions, to standardize retention behaviour on the user's columns, to monitor column performance and to compare packing materials. In the development of each prediction mode, experimental peak heights, peak widths, and retention times from model synthetic peptide standards were incorporated directly into the program and can be used as default values. Pro Digest-LC is an interactive program, in that researchers can run peptide standards on their particular columns and enter the peak width at half-height, peak height, retention time and quantity injected to adjust the simulation to their particular column. The simulated experiments eliminate the time-consuming trial-and-error methods used to suitable separation or purification procedures. The researcher would perform the actual experiment only after predicting the optimized conditions, thereby saving valuable sample and research time. The general concepts of the program along with representative separations of protein digests are displayed.