Protein phosphorylation is an important post-translational modification that regulates almost all cellular processes, such as cellular metabolism, growth, differentiation, signal transduction, and gene regulation. Mass spectrometry, which acts as an automated and sensitive method, enables global analysis of protein phosphorylation. However, several technical challenges need to be addressed when analyzing protein phosphorylation in a global manner. Low-abundant phosphopeptides need to be enriched before analysis with LC-MS/MS, so specific enrichment of phosphopeptides is central for a successful analysis of the phosphoproteome. Due to the complexity of phosphoproteome, fractionation of phosphopeptides before LC-MS/MS is essential to increase it coverage. Here, we present a detailed protocol for in-depth analysis of tissue phosphoproteome, including collection of tissue samples, extraction of tissue proteins, proteolytic digestion of proteins into peptides, enrichment of phosphopeptides with TiO2 using lactic acid as non-phosphopeptide excluder, fractionation of phosphopeptides with TEA-based high-pH reversed-phase (HpH-RP) chromatography, and identification of phosphopeptides with LC-MS/MS. We also outline the essential steps for data processing.