Antibiotics are secondary metabolites produced by microorganisms, extremely important to the health of our society. Cephalosporins are broad-spectrum antibiotics which are very similar in structure and action to penicillins but more resistant to ?- lactamases. Optimization of media is useful to increase the production of antibiotics. Induction of mutation is commonly employed to increase the yield of secondary metabolites like antibiotics. Chemical mutation is preferred method because of the ease in handling and avoiding the hazardous effects of radiations. Usually, mutations are induced in those microorganisms, which are well-known and well adapted for the production of that particular product. Selected microbes are cultured and treated with chemicals. The mutants are then screened for the particular product. There is an evidence of increasing production of secondary metabolites. The HPLC methods used in biotechnology are compared with other used for on-line analysis of ?-lactams in biotechnology. The data based on HPLC analysis is much reliable.