The xMAP food allergen detection assay (xMAP FADA) is an advanced multiplex immunoassay with multiple antibodies for each of 15 target food allergens and gluten, allowing for signal confirmation and antigenic profiling to occur in a single analysis. Botanicals used as spices are complex matrices for allergen analysis because they can exhibit inherent cross-reactivity with antibodies employed by the assays. Preliminary examinations of botanicals revealed chili peppers to have notably high levels of cross-reactivity with Brazil nut and hazelnut antibody bead sets in the xMAP FADA. This in-depth investigation of 29 pre-ground and whole chili peppers indicated Brazil nut and hazelnut cross-reactivity to be consistent among most members of genus, although cross-reactive signals generated by chili peppers were distinguishable from signals indicative of target allergen detection. Using the requirements that complementary antibodies used in the assay generated positive responses and that the various secondary end points were characteristic of the target analytes, xMAP FADA reactivity to chilis of the genus was categorized as cross-reactivity instead of confirmed detection of target allergenic foods.