Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient. It is expected to have broad applications in phosphoproteomics analysis.