The S. cerevisiae lysate DIA dataset that was acquired on a Q-Exactive using a 10-m/z-wide isolation windows DIA approach. The precursor mass range from 500 to 700 m/z is analyzed with twenty non-overlapping 10-m/z wide isolation windows targeted MS/MS scans. This dataset contained 6 biological replicates; each included peak boundaries of 204 manually curated peptides verified by DDA identification.