Protein labeling with a functional molecule is a technique widely used for protein research. The covalent reaction of self-labeling peptide tags with synthetic probe-modified small molecules enables tag-fused protein labeling with chemically diverse molecules, including fluorescent probes. We report the discovery, by in vitro directed evolution, of a novel 23-mer dibenzocyclooctyne (DBCO)-reactive peptide (DRP) tag using Systematic Evolution of Ligands by EXponential enrichment (SELEX) with a combination of a reconstituted cell-free translation system (PURE system) and cDNA display. The N- and C-terminal DRP truncations created a shorter 16-mer DBCO-reactive peptide (sDRP) tag without significant reactivity reduction. By fusing the sDRP tag to a model protein, we showed the chemical labeling and in-gel fluorescence imaging of the sDRP-fused protein using a fluorescent DBCO probe. Results showed that sDRP tag-mediated protein labeling has potential for use as a basic molecular tool in a variety of applications for protein research. (C) 2020 Elsevier Inc. All rights reserved.