Linezolid and voriconazole are two antimicrobials used for severe infections in critically ill patients. Pharmacokinetics and pharmacodynamics are altered in critically ill patients. Therefore, standard dosing of anti-infective agents may not reach the optimal therapeutic targets. A rapid and simple liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of linezolid and voriconazole in human serum only 3 min after one-step protein precipitation pretreatment to monitor their concentrations. Multiple-reaction monitoring (MRM) mode was used for detection. The calibration curves were linear over the range of 0.5–100 μg/mL for both linezolid and voriconazole, with regression coefficients above 0.9900 for all analytes. The intra- and interday coefficients of variation were below 15% at all concentration levels (LLOQ/LQC/MQC/HQC). This method was successfully applied to routine therapeutic drug monitoring (TDM) for critically ill patients and other patients in need.