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Background  It is not enough to optimize proteomics assays. It is critical those assays are robust to operating conditions. Without robust assays, proteomic biomarkers are unlikely to translate readily into the clinic. This study outlines a structured approach to the identification of a robust operating window for proteomics assays and applies that method to Sequential Window Acquisition of all Theoretical Spectra Mass Spectroscopy (SWATH-MS).    Methods  We used a sequential quality by design approach exploiting a fractional screening design to first identify critical SWATH-MS parameters, then using response surface methods to identify a robust operating window with good reproducibility, before validating those settings in a separate validation study.    Results  The screening experiment identified two critical SWATH-MS parameters. We modelled the number of proteins and reproducibility as a function of those parameters identifying an operating window permitting robust maximization of the number of proteins quantified in human serum. In a separate validation study, these settings were shown to give good proteome-wide coverage and high quantification reproducibility.    Conclusions  Using design of experiments permits identification of a robust operating window for SWATH-MS. The method gives a good understanding of proteomics assays and greater data-driven confidence in SWATH-MS performance.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12014-021-09323-z.

Robust optimization of SWATH-MS workflow for human blood serum proteome analysis using a quality by design approach