AbstractA method for extracting spiramycin by an octadecylsilica cartridge is described for plasma or vitreous samples. The macrolide antibiotic is then measured by reversed‐phase HPLC with UV detection. The limit of detection is estimated to be 50 ng/mL. The coefficient of variation for the procedure is 6.1% and 5.2% for the range of concentrations 0.2 μg/mL and 10 μg/mL respectively. By this method, pharmokinetic profiles were performed for five adult patients. Spiramycin could be accurately measured in the vitreous humour, allowing the determination of antibiotic at its site of act