Composition variation of a complex peptide mixture under enzymatic transformation can be tracked by mass spectrometry (MS). In this report, papain-catalyzed esterification of fibroin peptides was investigated at the individual peptide level using liquid chromatography-mass spectrometry with selected ion monitoring. Optimal conditions for maximizing ester formation were obtained using a water-to-pentanol ratio of 1:9 at pH 2.8 and $40^{circ}C$; however, the optimum conditions varied for individual peptides. The optimum pH levels were 2.5 and 2.8 for the tetrapeptides with a tyrosine or a valine residue and those with alanine or serine residues, respectively. The optimum pH shifted to 3.4 for dipeptide esters with a tyrosine residue. Tetrapeptides had a relatively higher rate of esterification above $50^{circ}C$. Alhough, the profiles of peptides and their esters in the esterification reaction were significantly affected by the reaction conditions, alanyl-glycine ester represented the largest fraction in the mixture under most reaction conditions. As demonstrated here, MS analysis of peptide mixtures can be used to elucidate specific reaction conditions for the enrichment of particular peptide products.