An analytical validation of a new liquid chromatographic-mass spectrometric (LC-MS/MS) method for simultaneous determination of metronidazole and spiramycin I concentrations in human plasma, saliva and gingival crevicular fluid (GCF) is presented. Ornidazole was used as an internal standard, and sample pre-treatment consisted of a liquid-liquid extraction. Chromatographic separation was achieved on a 5 microm Kromasil C18 column (150 mm x 4.6 mm i.d., particle size 5 microm), with a gradient using acetonitrile, water and formic acid at a flow rate of 0.9 ml/min. The methods were validated in terms of intra- and inter-batch precision (<7.1% in plasma, 12% in saliva and 9.2% in GCF, respectively) and accuracy (within +/-8.7% in plasma, within +/-8.7%, except LDQ level within +/-15.4% in saliva and within +/-10.7% in GCF), linearity, specificity, recovery (extraction efficiency), matrix effect, dilution process, stability in human plasma and saliva after three freeze-thaw cycles, stability in human plasma and saliva at ambient temperature and stability of the extracts in the automatic injector of the HPLC system. The methods are applicable for accurate and simultaneous monitoring of the plasma, saliva and gingival crevicular fluid levels of metronidazole and spiramycin I from pharmacokinetic studies.