A simple and precise ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) method was developed for the simultaneous analysis of five anti-infective agents used to treat severe infections [three antibiotics (daptomycin, moxifloxacin, ciprofloxacin) and two antifungals (isavuconazole, caspofungin)] in human plasma. Sample preparation was based on protein precipitation with ice cold methanol. All five agents were analyzed with the corresponding isotopically labeled internal standards. All analytes were detected in multiple reactions monitoring (MRM) using API 4000 triple-quadrupole mass spectrometer with electrospray (ESI) source operating in positive mode. The calibration curves were linear over the selected ranges (r>0.99). The method is precise and accurate with a total run time of 5.5min. Accuracy of all target analytes ranged between 95.9–116.6%, measured with an imprecision of less than 10.8%. The lower limit of quantification was 1.25mg/L for caspofungin, 0.3125mg/L for isavuconazole, 3.125mg/L for daptomycin, 0.075mg/L for ciprofloxacin, and 0.1875mg/L for moxifloxacin. The successful application of the method in patient samples proved its suitability for the medical surveillance of antimicrobial therapy in intensive care units as well as to other pharmacokinetic studies.