Rationale A derivatization switchable solvent liquid-liquid microextraction quadruple isotope dilution gas chromatography mass spectrometry (D-SS-LLME-ID4-GC/MS) method is presented for the determination of hydroxychloroquine sulfate in human biofluids. Methods While mixing type/period and concentration of NaOH were optimized via a univariate optimization approach, a multivariate optimization approach was used to determine optimum values for relatively more important parameters such as volumes of derivatization agent (acetic anhydride), NaOH and switchable solvent. Results Under the optimum experimental conditions, limit of detection and limit of quantification were calculated as 0.03 and 0.09 mg/kg (mass based), respectively. An isotopically labelled material (hydroxychloroquine methyl acetate-d(3)) was firstly synthesized to be used in ID4 experiments which give highly accurate and precise recovery results. After the application of D-SS-LLME-ID4, superior percent recovery results were recorded as 99.9 +/- 1.6-101.3 +/- 1.2 for human serum, 99.9 +/- 1.7-99.8 +/- 1.8 for urine and 99.6 +/- 1.5-101.0 +/- 1.1 for saliva samples. Conclusions The developed D-SS-LLME-ID4-GC/MS method compensates the complicated matrix effects of human biofluids and provides highly accurate quantification of an analyte with precise results.