Cactus seeds have unusual properties that resist protein extraction, e.g. a thick testa (seed coat) and a high amount of mucilage within the cells. This study was performed to determine which method (phenol extraction, TCA/acetone extraction, or phosphate-TCA/acetone extraction) is optimal for extracting protein from seeds of three species of Opuntia: Opuntia robusta, Opuntia phaeacantha, and Opuntia engelmannii. Extracted protein was analyzed using a Bradford assay and SDS-PAGE with silver staining. Overall results revealed that the TCA/acetone and phosphate-TCA/acetone methods were far more effective at extracting protein compared to the phenol extraction method. These protein extraction results can be applied to future studies of proteins important for Opuntia seed germination and conservation.