BACKGROUND: Hydroxychloroquine (HCQ) is approved for the treatment of systemic lupus erythematosus (SLE). Therapeutic drug monitoring (TDM) of HCQ is necessary to detect non-adherence and to improve treatment efficacy in SLE patients. Liquid chromatographic-tandem mass spectroscopy and HPLC-fluorescent methods are currently used to measure whole blood concentrations of hydroxychloroquine and its two main metabolites desethylhydroxychloroquine (DHCQ) and desethylchloroquine (DCQ) in SLE patients. This study reports the development and validation of an ultra-high performance liquid chromatography (UHPLC) method with fluorescence detection for the simultaneous quantification of HCQ and its metabolites in whole blood. METHODS: After adding chloroquine (internal standard) to the samples, a single-step protein precipitation and a subsequent filtration were employed for blood sample preparation. Analytes were separated under isocratic elution on a U-HPLC RP18 column with a total run time of seven minutes. The mobile phase consisted of piperazine buffer (46.4 mM, pH=9.8) and acetonitrile (68:32, v/v), was delivered at a flow rate of 0.4 mL/min. Fluorescence excitation and emission wavelengths were 335 and 390 nm, respectively. Assay performance parameters were evaluated per FDA bioanalytical guidelines. RESULTS: The calibration curve was linear from 125 to 4000 ng/mL for HCQ. The lower limit of quantification was 10 ng/mL for all analytes. For HCQ, DCQ and DHCQ, accuracies and imprecisions ranged from -7.90 to 7.85% and 1.14 to 8.78%, respectively. CONCLUSION: A sensitive, accurate and fast U-HPLC-fluorescent method was validated and successfully applied to quantify whole blood concentrations to perform TDM of HCQ in pediatric and adult lupus patients.