Collision cross section (CCS) of peptide ions provides an important separation dimension in liquid chromatography/tandem mass spectrometry-based proteomics that incorporates ion mobility spectrometry (IMS), and its accurate prediction is the basis for advanced proteomics workflows. We constructed an experimental peptide CCS dataset using phosphoproteome data. To obtain a unique set of peptides, we digested HeLa cell extracts with seven proteases (trypsin, LysargiNase, Lys-C, Lys-N, Glu-C, Asp-N, and chymotrypsin), enriched phosphopeptides from the digests, and fractionated the resulting phospho peptides with SCX-StageTip. We then dephosphorylated the phosphopeptides using calf intestine alkaline phosphatase to generate non-phosphorylated peptides with more missed cleavages.