Sulfotransferase enzymes (SULT) catalyse sulfoconjugation of drugs, as well as endogenous mediators, gut microbiota metabolites and environmental xenobiotics. To address the limited evidence on sulfonation activity from clinical research, we developed a clinical metabolic phenotyping method using paracetamol as a probe substrate. Our aim was to estimate sulfonation capability of phenolic compounds and study its intraindividual variability in man. A total of 36 healthy adult volunteers (12 men, 12 women and 12 women on oral contraceptives) received paracetamol in a 1 g-tablet formulation on three separate occasions. Paracetamol and its metabolites were measured in plasma and spot urine samples using liquid chromatography-high resolution mass spectrometry. A metabolic ratio (Paracetamol Sulfonation Index-PSI) was used to estimate phenol SULT activity. PSI showed low intraindividual variability, with a good correlation between values in plasma and spot urine samples. Urinary PSI was independent of factors not related to SULT activity, such as urine pH or eGFR. Gender and oral contraceptive intake had no impact on PSI. Our SULT phenotyping method is a simple non-invasive procedure requiring urine spot samples, using the safe and convenient drug paracetamol as a probe substrate, and with low intraindividual coefficient of variation. Although it will not give us mechanistic information, it will provide us an empirical measure of an individual's sulfonator status. To the best of our knowledge, our method provides the first standardised in vivo empirical measure of an individual's phenol sulfonation capability and of its intraindividual variability. EUDRA-CT 2016-001395-29, NCT03182595 June 9, 2017.