Twenty-three o-phthalaldehyde-mercaptoethanol derivatives of primary amino acids in serum were separated with Waters C18 5-microns Radial-Pak Resolve columns, using aqueous phosphate-methanol mobile phase with acetate and tetrahydrofuran as modifiers. Resolution is critical in this system for glutamine/histidine, citrulline/glycine/threonine/3-methylhistidine, and tryptophan/methionine derivatives, and is affected by small changes in column properties or mobile phase. However, pre-run adjustments in gradient scheme and/or mobile phase composition can usually be used to obtain chromatograms in which all derivatives can be quantitated. For this purpose, and for general method development, we have written two very fast, interactive programs for the Zenith Z-100 Microsoft BASIC compiler: RTGRAPH, for retention, separation, and resolution plots; and LCSIM for simulations of multisegment binary gradient elution. These programs are shown here to be useful and accurate in most aspects required for developing strategies for this method.