Updated project metadata. One of the major additions in mass spectrometry technology has been the irruption of the Orbitrap mass analyzer, which has boosted the proteomics analyses of biological complex samples since its introduction. Here we assessed the performance of the new generation Orbitrap Fusion Lumos Tribrid mass spectrometer in the identification and quantitation of peptides and phosphopeptides in single-shot analyses of human whole cell lysates using robust data-dependent acquisition methods. Our study explored the capabilities of the instrument for (phospho-)peptide identification and quantitation using different gradient lengths, different sample amounts, and different combinations of peptide fragmentation and mass analysers acquisition methods. Moreover, the acquisition of the same complex sample with different acquisition methods resulted in the generation of a dataset to be used as a reference for further analyses, and a starting point for future optimizations in particular applications.