Data-independent acquisition (DIA) strategies coupled with Evosep chromatography have recently gained popularity in achieving a high-throughput and robust mass spectrometry-based proteomics, but with compromised peptide identifications leading to lower peptide to protein ratios. Since premade gradient methods fix the flow rate and gradient length in the Evosep, a limited number of analytical columns fitting pressure/flow profiles can be used for peptide separation. To circumvent this limitation, we evaluated and standardized the performance of different dimensions of analytical columns with the Evosep and compared it with an EasyLC using a timsTOF-MS instrument in diaPASEF mode. The results reveal optimized usage of a specific column dimension that achieves higher sensitivity and deep proteome coverage