Mycoplasma genitalium is an important pathogen in male nongonococcal urethritis (NGU). Isolation of M. genitalium from clinical specimens by axenic culture is very difficult and time-consuming, and very few strains are available for antibiotic susceptibility testing. Primary isolation of M. genitalium by coculture with Vero cells improves the isolation rate significantly. However, some strains cannot be adapted to axenic culture. In this study, we determined the antibiotic susceptibility of M. genitalium strains grown in Vero cell culture with dilutions of antibiotics. Growth of M. genitalium was monitored by a quantitative PCR assay detecting a single-copy region of the mgpB adhesin gene. Growth inhibition in the presence of antibiotics was expressed as a percentage of the DNA load of controls grown in the absence of antibiotics. Eighteen strains were examined, including 6 new strains isolated from urethral swab specimens and 4 new strains isolated from urine specimens collected from Japanese men. Eight strains adapted to axenic culture were also tested by the conventional broth dilution method. The two methods had an acceptable correlation. Azithromycin was the most active drug against M. genitalium. Among the fluoroquinolones, moxifloxacin had the highest activity, with MICs ranging from 0.03 to 0.5 mg/liter, whereas ciprofloxacin and levofloxacin were considerably less active, with MICs ranging from 0.5 to 16 mg/liter and 0.25 to 4 mg/liter, respectively. MICs for tetracycline ranged from 0.125 to 4 mg/liter. This new method could increase the number of M. genitalium strains available for antibiotic susceptibility testing and significantly shorten the time from sampling to MIC results.