Global phosphoproteome profiling can provide insights into cellular signaling and disease pathogenesis. To achieve comprehensive phosphoproteomic analyses with minute quantities of material, we developed a rapid and sensitive phosphoproteomics sample preparation strategy based on ultrasound. We found that ultrasonication-assisted digestion can significantly improve peptide identification by 20% due to the generation of longer peptides that can be detected by mass spectrometry. By integrating this rapid ultrasound-assisted peptide-identification-enhanced proteomic method (RUPE) with streamlined phosphopeptide enrichment steps, we established RUPE-phospho, a fast and efficient strategy to characterize protein phosphorylation in mass-limited samples. This approach dramatically reduces the sample loss and processing time: 24 samples can be processed in 3 h; 5325 phosphosites, 4549 phosphopeptides, and 1888 phosphoproteins were quantified from 5 μg of human embryonic kidney (HEK) 293T cell lysate. In addition, 9219 phosphosites were quantified from 1-2 mg of OCT-embedded mouse brain with 120 min streamlined RUPE-phospho workflow. RUPE-phospho facilitates phosphoproteome profiling for microscale samples and will provide a powerful tool for proteomics-driven precision medicine research.