The far-UV spectral window widely used for the conformational analysis of biomolecules is not easily covered with broad-band lasers. This has made it difficult to use circular dichroism (CD) spectroscopy to directly follow fast structure changes. By combining transient CD spectroscopy in the deep-UV with thioamide substitution, we demonstrate a method to overcome this difficulty. We investigated a dipeptide whose two carbonyl oxygen atoms were replaced by sulfur, red-shifting the strong lowest-lying ππ* transitions into the more accessible 250–370 nm spectral window. Coupling of the two thioamide units cannot be resolved by achiral 2D-UV spectroscopy, but it gives rise to a pronounced bisignate CD spectrum. The transient CD spectra reveal weakening of this coupling in the electronically excited state, where conformational constraints are released. Our results show that direct local probing of fast backbone conformational change via CD spectroscopy is possible in combination with site-selective thio substitution in peptides and proteins.