S-Nitrosylation is a ubiquitous signaling process in biological systems. Research regarding this signaling has been hampered, however, by assays that lack sensitivity and specificity. In particular, iodine-based assays for S-nitrosothiols (1) produce nitrosyliodide, a potent nitrosating agent that can be lost to reactions in the biological sample being studied; (2) require pretreatment of biological samples with several reagents that react with proteins, artifactually forming or breaking S-NO bonds before the assay; and (3) are not sensitive or specific for nitrogen oxides in biological samples, reporting a wide range of different concentrations and falsely reporting NO-modified proteins, to be nitrite. These data, therefore, suggest that iodine-based assays should never be used for biological S-nitrosothiols. There are other assays that provide reasonably sensitive and accurate data regarding biological S-nitrosothiols, including assays based on mass spectrometry, spectrophotometry, chemiluminescence, fluorescence, and immunostaining. Each assay, however, has limitations and should be quantitatively complemented by separate assays. Continued improvement in assays will facilitate improved understanding of S-nitrosylation signaling.