Enrofloxacin is used for treating Shigellosis in non-human primates; however, there are no reports describing its pharmacokinetics in rhesus monkeys. Pharmacokinetic data in intended target species (rhesus) help to determine the proper dose regimen. Blood levels of enrofloxacin and ciprofloxacin (enrofloxacin's active metabolite), were determined after either intramuscular or oral dosing of enrofloxacin for 7 days in a cross-over study. Levels of both antibiotics were determined by solid phase extraction followed by reversed-phase chromatography with tandem mass spectrometry. Results indicate enrofloxacin half-life after intramuscular dosing is estimated to be 2.4 hours. Enrofloxacin given either intramuscular or p.o. rapidly achieves satisfactory therapeutic blood levels of enrofloxacin or ciprofloxacin in rhesus monkeys. Results from these pharmacokinetic study parallel values published for other animal species. Our results show use of enrofloxacin is effective in managing Shigella infections in rhesus monkeys based upon achieving these blood drug levels.