A selective, highly sensitive fluorimetric method is described for the determination of three α-aminocephalosporins, namely cephalexin, cefaclor and cephradine. Other cephalosporins free from the amino group did not interfere with the assay. The proposed method involves acid-hydrolysis of the drugs and subsequent alkalinization before measurement. The different experimental parameters affecting the fluorescence intensity were carefully studied and incorporated into the procedure. The method permits the determination of 0.02–0.40, 0.04–0.60 and 0.08–0.80 ug.ml−1for cephalexin, cefaclor and cephradine respectively with minimum detectability of 0.2 ng.ml−1for all cephalosporins. The proposed method was successfully applied to the determination of these drugs in formulations and biological fluids. The advantages of the described method over other existing methods were discussed. A route for the reaction pathway proposed.