Our previous review on the liquid chromatographic (LC) analysis of anti-bacterial agents was published in 1990 in a special issue of the Journal of Chromatography. Eight years later, some new agents have been registered and numerous other are under clinical experiment. In spite of therapeutic problems encountered with certain bacterial pathogens, the development of novel drug candidates has slowed partially due to the need for identification of new bacterial targets and the cost of the research. The present overview updates the LC methods for the quantitations of recent antimicrobial agents (marketed and in clinical development) in human biological fluids. Consideration has been given to procedures permitting the determination of isomers and metabolites as well as methods regarding tissue extracts or liquid sampled from physiological sanctuaries. LC methods are available for the quantitation of almost all registered or investigated recent anti-infective drugs and some are applicable in routine practice. Nevertheless, few techniques have been validated for the determination in tissue extracts limiting the development of penetration studies.