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Spot urine samples were analyzed 6 h after 200 mg caffeine consumption, following a 30 h methylxantine\u2010free diet. The major urinary caffeine metabolites are 1\u2010methyluric acid (1U), 5\u2010acetylamino\u20106\u2010formylamino\u20103\u2010methyluracil (AFMU), 1\u2010methylxanthine (1X), 1,7\u2010dimethyluric acid (17U) and 1,7\u2010dimethylxanthine (17X). CYP1A2, CYP2A6, XO and NAT\u20102 activities were estimated from the metabolic ratios (AFMU + 1U + 1X)\/17U, 17U\/17X, 1U\/(1X + 1U) and AFMU\/(AFMU + 1U + 1X), respectively. Metabolites and internal standard were extracted with chloroform\/isopropanol (85:15, v\/v) and separated on a C<jats:sub>18<\/jats:sub> column by an isocratic HPLC system using a two\u2010step elution with manual switch from solvent A (0.1% acetic acid\u2013methanol\u2013acetonitrile, 92:4:5 v\/v) to solvent B (0.1% acetic acid\u2013methanol, 60:40, v\/v), and detected at 280 nm. The method exhibited adequate metabolite separation (resolution factors &gt;1.48), accuracy (94.1\u2013106.3%) and intraday and interday precision &lt;8.02 and &lt;8.78%, respectively (<jats:italic>n<\/jats:italic> = 6). Smoking affected only CYP1A2, whereas gender had no effect in any enzyme activity. NAT\u20102 exhibited bimodal distribution, 63.6% of volunteers being slow acetylators. The developed RP\u2010HPLC method was fully validated and successfully applied for the evaluation of CYP1A2, CYP2A6, XO and NAT\u20102 activities. 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