Aims: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are minor metabolites of ethanol, and their presence in urine provides a strong indication of recent alcohol administration. In this study, we performed a drinking experiment to investigate the kinetics of EtG and EtS formation and elimination after the administration of two doses of alcohol. Methods: Nineteen volunteers provided urine and serum (only 18) after administration of 4 and 8 units of alcohol (1 unit corresponds to 10 ml or ∼8 g of pure ethanol). The analysis was performed using a validated ultra-performance liquid chromatography-mass spectrometry (UPLC®-MS/MS) method. Results: After 4 units, the median EtG maximum concentration (Cmax) was 0.4 µg/ml and the interquartile range (0.3 µg/ml) in serum and 3.5 mg/h (1.2 mg/h) in urine and were reached (Tmax) after 2.0 h (0.8 h) and 3.0 h (1.0 h), respectively. EtS Cmax was 0.2 µg/ml (0.1 µg/ml) in serum and 1.3 mg/h (0.6 mg/h) in urine, and the corresponding Tmax were 1.0 h (1.0 h) and 2.0 h (0.5 h). After 8 units, EtG Cmax was 1.3 µg/ml (0.4 µg/ml) in serum and 10 mg/h (3.4 mg/h) in urine and was reached after 4.0 h (1.8 h) and 4.0 h (2.0 h), respectively. EtS Cmax was 0.6 µg/ml (0.1 µg/ml) in serum and 3.5 mg/h (1.1 mg/h) in urine, the corresponding Tmax were 3.0 h (1.0 h) and 3.0 h (1.0 h). The EtG/EtS ratio increased as a function of the time after alcohol administration in both serum and urine samples but to a lesser extent after 8 units than 4. Conclusion: These results correlate with values obtained in previous studies. Tmax of EtG and EtS increased between 4 and 8 units. The EtG:EtS ratio increased in the serum and urine samples of all volunteers as a function of time at least up to 4 h after alcohol administration.