The modulation of peptidic scaffolds through stapling reactions has been established as a powerful tool for recapitulating the bioactivity of native ⍺-helices in targeting protein-protein interactions (PPIs). However, accessing such helices have largely relied on protecting group manipulations or the use of non-natural building blocks during peptide synthesis. As such, there is a growing need for a stapling strategy involving only natural amino acids in their unprotected states. Herein we report a rapid, mild, and highly chemoselective stapling reaction using a new class of molecular linchpins called 2-ketobenzaldehydes that installs a highly fluorescent thiol-isoindole crosslink. This methodology also exhibited good positional selectivity favoring the helical i and i+4 linkage on fully unprotected peptides in the presence of competing reaction sites, offering exceptional late-stage functionality for easier access of stapled ⍺-helices. In our efforts to further validate this chemistry, we have successfully shown in vitro cytotoxicity (IC₅₀ = 5.10 µM) equipotent to an all-hydrocarbon stapled peptide candidate. Furthermore, in harnessing the innate fluorescence of thiol-isoindole, this staple can be directly used as a probe for cell imaging in the qualitative assessment of stapled-peptide cell permeability, thus bridging therapeutic potential with analytical probe development.