Data acquisition workflow to generate a comprehensive E. coli assay library, quality evaluation with DIALib-QC and DIA/SWATH-MS quantification by Spectronaut. A comprehensive DIA/SWATH assay library for E. coli was generated from whole cell lysate, fractionated samples, overexpressed proteins, and supplemented with synthetic peptides. Samples were analyzed with data-dependent acquisition (DDA) mass spectrometry on TripleTOF 5600+ and TripleTOF 6600 instruments resulting in 209 data files. To generate a DIA/SWATH library, the raw data files were converted to mzML format using the ABSCIEX converter with the profile mode extraction parameter. The mzML files were searched against the reference proteome using both Comet and X!Tandem search engines. The identified sequences were then statistically validated using the Trans-Proteomic Pipeline (TPP) including PeptideProphet and iProphet. MAYU was applied to control the FDR at the protein level. Using SpectraST, confidently assigned spectra were converted into a redundant spectral library and retention times are normalized in iRT space using RTCatalog, then a consensus spectrum library was generated. The assay library was extracted from the consensus library using the spectrast2tsv.py script. Libraries were evaluated with the DIA Library Quality Control (DIALib-QC, www.swathatlas.org) tool and their assessment reports were generated. The performance of the TripleTOF E. coli spectral library was evaluated based on the identification and quantitation of peptides and proteins in data-independent acquisition (DIA) methods with different gradient lengths using the Spectronaut analysis software.