INTRODUCTION: No information exists on therapeutic or toxic concentrations of tetrahydrozoline, which has been reported to be used in drug facilitated sexual assaults. The primary aim of this investigation was to establish baseline therapeutic serum and urine concentrations in a sample of healthy volunteers. METHODS: Ten healthy volunteers consented to have two drops of Original Visine(R) eye drops (0.05% tetrahydrozoline solution) placed directly into the conjunctival sac of each eye, 30 s apart, at times 0, 4, 8, and 12 h. Blood and urine samples were then collected at 2, 5, 9, 13, and 24 h post-application and analyzed for concentrations. Tetrahydrozoline concentrations are described using measures of central tendency and dispersion at each time point, with predictions of serum and urine concentrations over time calculated using a linear mixed effects regression model. RESULTS: Tetrahydrazoline concentrations were detectable in both serum and urine after therapeutic ocular administration. The mean serum half-life of tetrahydrozoline was approximately 6 h. Systemic absorption varied among subjects, with the maximum serum concentrations ranging from 0.068 to 0.380 ng/ml. At 24 h, all patients had detectable urine concentrations of tetrahydrozoline (range = 13?210 ng/ml). CONCLUSION: When used as directed by the manufacturer for therapeutic ocular administration, tetrahydrozoline concentrations were detectable in both serum and urine up to 12 h after the last administered dose. A concentration greatly exceeding the 95% confidence interval of drug present during therapeutic ocular use may be suggestive of illegal adulterant use or accidental or suicidal overdose.