Clindamycin is widely used in antimicrobial prophylaxis to prevent surgical site infections. Adequate subcutaneous free tissue concentrations should reach therapeutic levels, which have to be maintained throughout the surgical procedure for antibiotic prophylaxis to be efficient. A method was developed and validated using high performance liquid chromatography coupled to a mass spectrometry to determine clindamycin concentrations in two biological matrices: plasma, for drug monitoring, and subcutaneous microdialysate, to determine free concentrations at the incision site. Gradient separation of clindamycin was carried out using a reverse phase C18 column eluted with a mixture of mobile phases (1% formic acid in water and 1% formic acid in acetonitrile). The monitored transitions were m/z 425.3 > 377.3 for clindamycin, and m/z 407 > 359 for lincomycin, used as the internal standard for plasma samples. Linearity was reached in the 0.5–100 μg/mL range for plasma and 25–1000 ng/mL for microdialysate samples. The method was selective, precise, and accurate, and was successfully employed in a preliminary pharmacokinetics study to investigate plasma and subcutaneous clindamycin penetration, determined by microdialysis, after intravenous administration of a 50 mg/kg dose to Wistar rats.