In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10(7) down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti(4+), our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research.