Technical Abstract: Red imported fire ants, Solenopsis invicta Buren, build nests by excavating soil. Incorporation of ant-derived chemicals in nesting material has long been known, however only a few chemicals have been identified. This paucity of identified ant-derived chemicals may be due to the interference from soil-borne compounds in chemical analysis. Red imported fire ants were able to build their nest using moistened silica gel as the only building material under the laboratory condition. This provided an opportunity to establish a profile of ant-derived chemicals in nest material without the presence of any soil borne interference chemicals. A new method for profiling ant-derived chemicals in nest material using GC-MS was developed. All samples contained cuticular hydrocarbons and venom alkaloids. Phosphoric acid, glycerol, lactic acid and malonic acid were identified from samples collected inside the nest.