Evaluation of immune mechanisms responsible for control of viral replication is critical to understanding HIV-2 attenuated biological characteristics in pathogenesis and transmission. Evaluation of the cellular immune response is often based on labor-intensive techniques that limit the scope of most studies performed. A simple and rapid anthrax toxin-based ELISPOT method to assess HIV-2 cellular immune response was developed. The modified anthrax toxin-based antigen presentation process performed better than a recombinant vaccinia system and the ELISPOT method significantly enhanced the ease and simplicity of the assay. Using this method, a robust HIV-2 cellular immune response directed toward the p26 core protein was exhibited in 21 of 24 (87.5%) infected women, and all 8 seronegative subjects were negative in both assays. Cellular immune responses were associated with low HIV-2 viral load. This simple and rapid modified anthrax toxin-based ELISPOT method allowed us to demonstrate, strong cellular immune responses that may be critical determinants in the HIV-2 attenuated phenotype.