The development of a supercritical fluid extraction procedure for the extraction of clenbuterol from bovine liver tissue is described. The procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol residues at the low ppb level. Method validation incorporating inter- and intra-assays was carried out on fortified liver tissue and showed good recoveries and low variations (RSD < 15%) for levels of clenbuterol of 0.5, 2 and 5 ppb. The developed procedure was shown to be successful for the determination of clenbuterol in incurred liver tissue. The effects of organic modifiers and of inherent sample moisture on analyte extractability are also presented.