Co-trimoxazole is frequently used in the prophylaxis and treatment of Pneumocystis carinii pneumonia. High plasma concentrations of sulfamethoxazole or trimethoprim are correlated with toxicity. There is, however, a large variation in PK observed which can lead to underexposure or toxicity. We developed a novel LC-MS/MS method to analyze the components of co-trimoxazole, trimethoprim and sulfamethoxazole and its metabolite sulfamethoxazole-N-acetyl. This new method is expeditious due to its limited sample preprocessing and a relatively short run-time of only 3 min. This new method met the US FDA requirements on linearity, selectivity, precision, accuracy, matrix effects, recovery and stability and is suitable for routine analysis and future prospective studies.