Here we describe the use of data independent acquisition (DIA) on a Q-Exactive mass spectrometer for the detection and quantification of peptides in complex mixtures using the Skyline Targeted Proteomics Environment (freely available on-line at http://skyline.maccosslab.org). The systematic acquisition of MS/MS spectra by DIA is in contrast to DDA where the acquired MS/MS spectra are only suitable for identification of a stochastically sampled set of peptides. Similar to selected reaction monitoring (SRM), peptides can be quantified from DIA data using targeted chromatogram extraction. Unlike SRM, data acquisition is not constrained to a pre-determined set of target peptides. In this protocol, a spectral library is generated using data dependent acquisition (DDA), and chromatograms are extracted from the DIA data for all peptides in the library. Similar to SRM, quantification using DIA data is based on the area under the curve of extracted MS/MS chromatograms. Additionally, a quality control method suitable for DIA based on targeted MS/MS acquisition is detailed. Not including time spent acquiring data, and time for database searching, the procedure takes about 1–2 hours to complete. Typically, data acquisition requires roughly 1–4 hours per sample and a database search will take 0.5–2 hours to complete.