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We review 50 years of use of 2′,3′-O-trinitrophenyl (TNP)-ATP, a fluorescently tagged ATP analog. It has been extensively used to detect binding interactions of ATP to proteins and to measure parameters of those interactions such as the dissociation constant, Kd, or inhibitor dissociation constant, Ki. TNP-ATP has also found use in other applications, for example, as a fluorescence marker in microscopy, as a FRET pair, or as an antagonist (e.g., of P2X receptors). However, its use in protein binding studies has limitations because the TNP moiety often enhances binding affinity, and the fluorescence changes that occur with binding can be masked or mimicked in unexpected ways. The goal of this review is to provide a clear perspective of the pros and cons of using TNP-ATP to allow for better experimental design and less ambiguous data in future experiments using TNP-ATP and other TNP nucleotides.