In this study, we designed primers and probes for the rpoB gene of Neisseria meningitidis to detect rifampicin-resistant strains in a combined use with primers and probes previously described for penicillin intermediate isolates. The assay was set up in the Light Cycler instrument using the Fluorescence Resonance Energy Transfer platform. The method was applied to N. meningitidis strains and to culture-negative cerebrospinal fluids (CSFs) from patients with meningococcal invasive disease. A spiked CSF was used to determine the sensitivity of the assay.