OBJECTIVE To investigate the utility of metrics of CYP1A2 activity using caffeine as a probe, and saliva and plasma sampling with or without a 24-h caffeine abstinence. METHODS This was a cross-over pharmacokinetic study in 30 healthy male subjects who received a single oral 100mg caffeine dose after 24-h caffeine abstinence or after maintaining their regular caffeine intake (no caffeine abstinence). Serial blood and saliva samples were collected simultaneously over 24h. Caffeine and paraxanthine concentrations were measured using a validated HPLC assay. RESULTS There was a strong correlation between the paraxanthine/caffeine AUC(0-24) ratio (reference metric) and the paraxanthine/caffeine concentration (C(t) ) ratio at 4h (C(4) ) in both saliva and plasma (r≥0.75). The paraxanthine/caffeine AUC(0-24) ratio in plasma and saliva did not differ between the 24-h caffeine abstinence and the no abstinence period (P>0.05). The optimal paraxanthine/caffeine C(t) that correlated with the plasma paraxanthine/caffeine AUC(0-24) ratio in the 24-h abstinence period was 2 and 4h (r=0.88) in plasma, and 4 and 6h in saliva (r=0.70), while it was the saliva 4h time-point in the no abstinence period (r=0.78). CONCLUSIONS The saliva paraxanthine/caffeine concentration ratio at 4h was a suitable metric to assess CYP1A2 activity after oral administration of caffeine without the need for 24-h caffeine abstinence.