Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2', 7'-Difluorofluorescein Diacetate (DAF-FM diacetate). This compound diffuses into cells and intracellular cleavage by esterase enzymes liberates weakly-fluorescent DAF-FM, which reacts with NO or other specific RNS to become highly fluorescent (Kojima et al., 1999). Although quantification of fluorescence is performed using a fluorescent plate reader, it is envisioned that this protocol could be adapted for intracellular NO/RNS imaging of S. aureus biofilms by confocal microscopy. Likewise, this technique could be optimized for the detection of intracellular NO/RNS in other growth conditions (i.e., planktonic cultures) and/or in other bacteria/archaea.