Phosphorylation, one of the most common post-translational modifications of proteins, plays a critical role in many biological processes. We have previously developed several analytical methods for determining the phosphorylation status of certain proteins by using a phosphate-capturing binuclear metal complex known as Phos-tag. Here, we describe a novel method for the gel-based in vitro analysis of the phosphorylation status of a protein by a simple and rapid fluorometric staining method that uses a tetramethylrhodamine (TAMRA)-labeled Phos-tag derivative (TAMRA-Phos-tag). The entire staining protocol, which requires less than 2 h to complete, uses three buffer solutions for staining, washing, and dilution, respectively, at room temperature. The gel-based analysis of phosphoproteins in a polyacrylamide gel can be conducted by using a fluorescence imaging scanner with a 532-nm excitation laser and a 580-nm longpass emission filter. As a practical example of the use of the TAMRA-Phos-tag staining method, we examined the time course of dephosphorylation of ovalbumin by an alkaline phosphatase. In addition, inhibitor profiling of a tyrosine kinase Abl was performed by using an Abl-substrate (GST-Abltide) and an Abl-inhibitor (Imatinib).