AIM: To determine a species-specific real-time polymerase chain reaction (PCR) assay to detect Pseudomonas aeruginosa (PA), a secondary DNA target for PA that may provide a universal target for other bacterial pathogens, and validate both assays for diagnostic testing. METHODS: PCR detection was established against the ecfX PA gene and the 16S rRNA gene using known PA keratitis isolates. The outcome parameters for both assays were “limit of detection” (LOD), amplification efficiency (AE), and PAGE amplified product analysis. Both assays were validated against 20 true-positive clinical samples positive for PA DNA and 20 true-negative samples containing no PA DNA. Descriptive statistics and PAGE analysis were used as outcome parameters. RESULTS: AE of the ecfX assay was 96.6%, and LOD was 33.6 copies of target DNA per microliter. AE of the 16S rRNA assay was 103.4%, and LOD was 8.12 copies per microliter. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency for the ecfX and 16S rRNA assays were [75%, 95%, 94%, 79%, and 85%], and [70%, 100%, 100%, 77%, and 85%], respectively. Both PCR assays were validated, followed by confirmation of DNA patterns from PAGE analysis. CONCLUSION: The PCR methodology described here may be a useful adjunct to standard methods in the diagnosis of PA keratitis.