We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe3+ metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.