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ABSTRACTHaemophilus influenzaeis a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzaediagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzaein RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpBgene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilusspp. Subsequently, 44 Haemophilusclinical isolates were collected, and 36 isolates were identified as H. influenzaeusing a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucKdiagnostic assay. Using the novel smpBdiagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n= 98) were blindly tested with the gold standard and smpBdiagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpBassay when used directly on respiratory specimens.