The dataset contains the data of key experiments for developing a novel and highly sensitive bottom-up phosphoproteomics strategy termed ERLIC-SCX/RP-LC-MS. We compared two SPE types (RP, SCX) for an ERLIC run with 7 fractions. The results were used to design a workflow for highly sensitive bottom-up phosphoproteomics (third experiment) combing ERLIC in 21 fractions with the fractions 1-9 cleaned by SCX-SPE and the fractions 10-21 by RP-SPE. Up to 50% of the fractions were subjected to LC-MS/MS analysis with 45 h of total MS time per replicate.