A simple and quick protocol for chemical treatment, enzymatic digestion, and subsequent identification of proteins on ProteinChip arrays is presented. Chicken ovalbumin, bovine fetuin and a heavily posttranslationally modified protein, human epidermal growth factor receptor extracellular domain, were employed as model proteins to evaluate the novel protocol. The chemical treatment includes denaturation, reduction, and alkylation, while enzymatic digestion encompasses deglycosylation, dephosphorylation, and digestion by various proteases. All reactions were carried out on-chip in a sequential fashion. Peptide mass fingerprinting identified several peptides derived from the three proteins. This protocol was also applied to the analysis of urinary proteins from a male rat with puromycin-induced proteinuria. r-2u-Globulin, the major urinary protein in the normal male rat and albumin, the most abundant in the treated animal, were readily identified. This procedure demonstrates that complete on-chip treatment can be used for rapid protein identification and structural characterization.