Post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation are an essential regulatory mechanism of protein function and they are associated with a range of biological processes. Since most PTMs alter the molecular mass of a protein, mass spectrometry (MS) is the ideal analytical tool for studying various PTMs. However, PTMs are generally present in substoichiometric levels and therefore their unmodified counterpart often suppresses their signal in MS. Consequently, PTM analysis by MS is a challenging task requiring highly specialized and sensitive enrichment methods. Currently, several methods have been implemented for PTM enrichment and each of them has its drawbacks and advantages as they differ in selectivity and specificity toward specific protein modifications. Unfortunately, for most of the more than 300 known modifications we have none or poor tools for selective enrichment.Here, we describe a comprehensive workflow to simultaneously study phosphorylation, acetylation, and N-linked sialylated glycosylation from the same biological sample. The protocol involves an initial titanium dioxide (TiO2) step to enrich for phosphopeptides and sialylated N-linked glycopeptides followed by glycan release and post-fractionation using sequential elution from immobilized metal affinity chromatography (SIMAC) to separate mono-phosphorylated and deglycosylated peptides from multi-phosphorylated ones. The IMAC flow-through and acidic elution is subsequently subjected to a next round of TiO2 enrichment for further separation of mono-phosphopeptides from deglycosylated peptides. In addition, the acetylated peptides present in the first TiO2 flow-through are enriched by immunoprecipitation (IP). Finally, the samples are fractionated by hydrophilic interaction liquid chromatography (HILIC) to reduce sample complexity and increase the coverage during LC-MS/MS analysis. This allows the analysis of multiple types of modifications from the same highly complex biological sample without decreasing the quality of each individual PTM study.