Abstract Four kinds of polyacrylamide gel electrophoresis (PAGE) were applied to insulin and peroxynitrite‐treated insulin. The Native‐PAGE had a better resolution than sodium dodecyl sulfate (SDS)‐PAGE, SDS‐urea‐PAGE, and even Tricine‐SDS‐PAGE. Reduction and nonreduction of insulin and peroxynitrite‐treated insulin in Native‐PAGE showed that four tyrosine residues in insulin molecular could be nitrated by peroxynitrite and that alkylation with iodoacetamide was better than no alkylation and alkylation with iodoacetic acid, which would introduce negative charges to the peptides. The method of Native‐PAGE was suitable to analysis of insulin and its analogs, even other peptides of low molecular weight.