A reversed-phase liquid chromatography with ultraviolet detection is described fr the determination of cotinine, 3-hydroxycotinine and also 3-hydroxycotinine/cotinine ratio. A mobile phase consisted of a mixture ammonium acetate:methanol (50:50). Using a CS-type column endcapping, a good separation was achieved in 8 mm. The wavelength of tJV detector was set at 260 nm. With acetanilide as internal standard, human urine samples were cleaned up by liquid-liquid extraction. The regression equations obtained from both the standard were linear between 1.0 - 5.0 ig/mL for 3-hydroxycotinine and between 2.0-10.0 .ig/mL (r> 0.999). This method has been successfully applied to the determination of urinary 3-hydroxycotinine and cotinine content and also for detennining 3-hydroxycotinine/cotinine ratio in several Javanese smokers from Indonesia.