As an example of an important redox-based protein posttranslational modification, protein S-nitrosation of specific cysteines is attracting more and more attention. The methods of detecting protein S-nitrosation in vitro or in vivo have been widely used in recent research, especially the biotin switch assay. An increase in band intensity in the presence of ascorbate is thought to be diagnostic for the presence of S-nitrosothiols. However, we found that this is a flawed assumption. In this study, bovine serum albumin (BSA) and even BSA prereduced by 20 mM 2-mercaptoethanol give false-positive signals for S-nitrosothiols (corresponding to a level of about 0.5–1% S-nitrosated BSA) when detected by the biotin switch assay. Higher blocking conditions could not diminish the signal, whereas omitting ascorbate in the step before biotinylation resulted in the disappearance of the signal. Further investigation of the mechanism showed that ascorbate increases the rate of the biotinylation reaction and accelerates the presence of the false-positive signal. Our results provide direct evidence that ascorbate could give rise to a significant false-positive signal in the biotin switch assay. Ascorbate treatment can interfere with the interpretation of the data. Hence, care should be taken when this method is used.