The red imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), produces venom that consists of alkaloids and a small amount of protein. The venom possesses a diversity of bioactivities. Several different techniques have been used for the extraction and analysis of fire ant venom. Here, we tested the quality of venom by comparing alkaloid profiles extracted by different methods such as capillary milking, venom gland dissection, solvent-soaking of ants without venom apparatus, and solvent-soaking of whole ants. The results indicated that whole body solvent-soaking extracts the same alkaloids that are found by dissection of the venom sac or those milked through the sting, suggesting that whole body solvent- soaking adequately extracts venom alkaloids from fire ant workers. Due to its simplicity and efficiency, venom extraction by solvent-soaking followed by conventional silica gel column chromatography can be used for isolating fire ant venom alkaloids for chemical analyses and bioassays.