ortho-Phthalaldehyde (OPA) and its derivatives are found to react chemoselectively with amino groups on peptides and proteins rapidly and tracelessly under the physiological condition via formation of phthalimidines, which provides a novel and promising approach when performing bioconjugation on native proteins. The notable advantages of this method over the existing native protein lysine-labeling approaches include a traceless process, a self-reacting, specific and fast reaction, ease of operation, and the ability to use nonhydrolyzable reagents. Its applications have been effectively demonstrated including conjugation of peptides and proteins, and generation of an active PEGlyated l-asparaginase.