Nanoflow liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) data was obtained with bioinertized systems for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only direct contact of phosphopeptides with metal components, but also indirect contact with nanoLC pumps through the mobile phase causes significant losses during the recovery of phosphopeptides. Therefore, metal ions were depleted by inserting an on-line metal ion removal device containing metal-chelating membranes between the gradient mixer and the autosampler.