Osteoclasts are the only bone-resorbing cells. In addition to other specific properties, osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), which is usually detected using a histochemical method for light microscopy. Using ELF97 phosphatase substrate, this study describes a new fluorescence-based method for TRAP detection. The fluorescence-based ELF97 TRAP stain not only results in a better resolution of the TRAP-positive granules, because confocal microscopy can be applied for image acquisition and analysis, but it reveals additional and more specific information about osteoclasts because it can be combined with other fluorescence-based methods.