A method involving protein precipitation with trichloroacetic acid and ampicillin hydrolysis with sodium hydroxide was developed for determination of ampicillin residue in milk and muscle. The hydrolysis product reacts in an acidic medium, in the presence of mercuric chloride, to form a fluorescent compound, which is extracted from the matrix with dichloromethane and concentrated on a silica Sep-Pak cartridge in a single step. After elution of the silica cartridge and evaporation of the solvent, the residue is recovered in 100 microL ethyl acetate, and 20 microL is deposited on a chromatoplate and chromatographed with ethyl acetate. The method allows determination of ampicillin residues in milk and muscle fortified at 4 micrograms/L and 50 micrograms/kg, respectively.