The principles and application of an in-vitro pharmacokinetic simulation system controlled by a stepwise method are described. In this system, drug concentrations in a culture medium are regulated by a periodic stepwise-adjustment method. The drug concentration is increased by adding a portion of concentrated drug solution, or decreased by draining a portion of the culture medium and the subsequent addition of drug-free medium. The drug concentration is adjusted each minute with peristaltic pumps controlled by a microcomputer. Concentrations of two drugs with independent pharmacokinetics can be simulated in the same medium. An algorithm for controlling drug concentrations by a microcomputer is presented in this report. This pharmacokinetic system was successfully applied to a urinary tract infection system, in which three infection models with different severity were reproduced in vitro. The in-vitro pharmacokinetic simulation system described here could be applicable not only for evaluating the bactericidal activities of antibacterial agents but also for evaluating other categories of drugs, such as antitumor agents, based on their pharmacokinetics.