OBJECTIVES: To develop a reliable ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for therapeutic drug monitoring (TDM) of unbound ciprofloxacin concentrations in critically ill patients. METHODS: Total and unbound ciprofloxacin concentrations of five randomly selected intensive care unit (ICU) patients were measured using UPLC-MS/MS. Method validation included accuracy, linearity, precision, repeatability, and limits of detection and quantification. RESULTS: The median unbound ciprofloxacin fraction was 74.8%, with a median area under the curve from 0-24 h (AUC0-24) and maximum serum concentration (Cmax) of 28.51 h·mg/L and 4.45 mg/L respectively. Median free AUC0-24 (fAUC0-24) and free Cmax (fCmax) were 21.57 h·mg/L and 3.53 mg/L respectively; 20% of patients reached the pharmacodynamic target. The UPLC-MS/ MS method was validated using an intra-assay and inter-assay precision < 3%. Recoveries were between 90-110% CONCLUSIONS: This UPLC-MS/MS method provided reliable unbound ciprofloxacin concentrations, allowing target attainment in critically ill patients and exploration of different dosing regimens.