A PCR based quantitative assay was used to determine Wolbachia infection levels in three different Drosophila strains. In addition, confocal microscopy was used to confirm and calibrate these results. Wolbachia infection levels ranged from 2,600 to 18,500 per egg. Single ovaries and testes from each of the three strains were also assayed using the calibrated quantitative PCR assay. A general correlation was found between bacterial levels in eggs and those found in ovaries and testis. These infection levels were consistent with the expression of cytoplasmic incompatibility (CI). In two strains of D. simulans, although the overall bacterial numbers were not significantly different, they exhibited different levels of CI. A direct correlation between the number of infected developing sperm cysts in these strains and CI levels was observed. This calibrated assay should provide a useful baseline for future comparative work, particularly between laboratories.