An automated tryptic mapping method was developed for characterization of disulfide linkages in recombinant human growth hormone (rhGH). The hormone was trypsin digested and the peptide fragments concentrated by eluting rhGH through an immobilized trypsin column and transferring the peptides directly to a reversed-phase liquid chromatography (RP-LC) column where they were collected. Reaction time was controlled by the flow-rate. Following tryptic digestion of a sample, the immobilized enzyme column was uncoupled from the flow train by a switching valve and the RP-LC column eluted with a solvent gradient ranging from 0.1% trifluoroacetic acid (TFA) with 1% acetonitrile (ACN) to ACN with 0.1% TFA and 5% water. This two-step mapping process was achieved within 2 h on both native and reduced rhGH samples. The chromatographic elution position and mass spectra matrix-assisted laser desorption ionization time-of-flight mass spectrometry of native rhGH and sulfur-containing peptides were determined with standards. Standards of the individual sulfhydryl (-SH) containing peptides and all possible disulfide linked peptides that could result from coupling the -SH peptides in disulfide linkages were obtained by synthesis and chromatographic purification. This approach allowed the chromatographic elution position of all possible mismatched disulfide containing peptides to be established and samples of rhGH to be examined for improper folding.