Elucidating protein conformations in vivo remains a formidable challenge in structural biology. A biochemical technique, disulfide scanning mutagenesis, has been used for decades to investigate in vivo protein structures, but it has been limited in practice by low throughput. We have developed high-throughput disulfide scanning (HTDS) by harnessing modern library-scale mutagenesis, deep sequencing, cysteine-targeted proteolysis, mass spectrometry, and atomistic structural modeling from disulfide distance restraints. We used HTDS to probe the conformational landscape of the periplasmic acid stress chaperone HdeA of E. coli, revealing both native and alternative conformations with distinct phenotypic effects. This study is a proof of concept that HTDS can directly link genotype to protein conformation and protein conformation to evolutionary fitness.