The biologically active natural and semisynthetic cephalosporin antibiotics require proper methods of extraction and purification for their isolation and subsequent pharmacological studies. This article reviews the various methods useful for extraction and purification of individual compounds as well as the enzymes involved in their biosynthesis. Applicability of the methods for downstream processing of the spent medium has been critically analysed. Adsorption chromatography, particularly with reverse phase materials, in combination with membrane separation is the most successful technique for extraction as well as purification of most of the enzymes and individual compounds. Techniques such as reactive extraction in liquid membrane, non-dispersive extraction in hollow fiber membrane and aqueous two-phase extraction are likely to emerge in new generation processes. Finally, some aspects of process design and scale-up have been discussed, highlighting the research needs of pragmatic importance.