Gluten is a commonly used cereal derivative found in bakery products, among other items. In some susceptible individuals, however, it triggers immune responses of different kinds; there is, to a lesser extent, the wheat allergy that is immunoglobulin E (IgE)-mediated and leads to histamine release and typical allergic symptoms. In this case, other water-soluble proteins, like albumins, are also involved. On the other hand, there is, more frequently, celiac disease (CD), where the gluten causes immune reactions in the intestines of certain individuals, leading to degeneration of villi, which typically leads to malabsorption of nutrients and, consequently, malnutrition. The only currently effective health strategy for affected consumers is avoidance of gluten-containing products, based on clear labeling rules. However, despite unanimously accepted Codex definitions by all member jurisdictions, the national implementation of equivalent laws shows significant differences. In the context of CD and in support of the gluten-free statement, regulatory enforcement, as well as manufacturers' quality controls are mostly based on analytical results. However, numerous methods are available, some of which have been validated better than others, and many provide different results on identical samples. Reasons include detection of different gluten components and variability in extraction efficiency due to different buffer compositions, especially from processed foods. Last but not least, the lack of reference materials is hindering the process of generating comparable data across different ELISA kits, as well as other methods. How can such data still be used to support a gluten-free claim? New methodologies, in particular mass spectrometric analysis of gluten derived peptides, are being introduced in numerous laboratories. This methodology is not only capable of detecting gluten derived peptides but can also differentiate between and quantitate wheat, barley, rye, and oat. This paper presents analytical limitations, as well as promising new approaches in support of industry and enforcement activities to ensure compliance with the gluten-free claim under the current regulatory framework.