We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and minimizes sample handling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.