Abstract: OBJECTIVE To establish a real-time PCR assay to fast detect bacterial contamination in drugs. METHODS Staphylococcus aureaus and Escherichia coli were used as representative, and the bacterial genomic DNA was isolated by lysis buffer kit and then quantitatively detected by Taqman real-time PCR. Propidium monoazide (PMA) was used as a pretreatment for the DNA molecules in dead cells and inhibited the PCR amplification of the DNA molecules. RESULTS PMA treatment was effective and the real-time PCR targeting conservative 16S rRNA gene showed high sensitivity. The Ct value of the lowest bacterial dose groups in both S. aureaus and E. coli were statistically different from the negative control with a minimal detection quality of 2 CFU/PCR. The result of real-time PCR was accordant to the pharmacopoeia test in the sterile test of contaminated drugs. CONCLUSION The lysis buffer kit used to extract bacterial genomic DNA with subsequent real-time PCR is convenient and sensitive, and may be applied to fast detection of drug sterility test.