Though a variety of chemical reactions of serine in natural systems has been known for decades, the challenges to achieve chemical modification of serines have been the longstanding issue of chemists, and the present work demonstrated the catalytic bioconjugation in biomolecule-compatible, nonaqueous acidic media. With the success of cell lysate labeling described above, the acylation method would be applicable to the profiling of the activity of serine residues that are not located in enzyme active sites. In contrast to the natural system facilitating reactions of a mildly reactive solvent molecule (i.e., water) through actions of enzymes as catalysts, the nonaqueous strategy promoted the acylation reaction with a solvent molecule through acid catalysis by reaching the extreme pH scale that would not be accessible in aqueous media. As acid catalysis is a common synthetic organic chemistry method, the acidic nonaqueous approach can be extended to a variety of other chemical transformations, which would expand the repertoire of the catalytic bioconjugation strategies.