Nicotine and cotinine in hair are good biomarkers for assessing long-term exposure to smoking. However, analytical devices such as GC/MS are associated with high cost and are not widely used. HPLC/UV is used widely in laboratories, but is unsuitable for measurement of minor con- stituents, except when using the column-switch- ing method. Thus, we aimed to establish a sim- ple, inexpensive and sensitive method based on HPLC/UV with column switching for measuring nicotine and cotinine in hair. First, we compared the presence and absence of a column selection unit. We then measured amounts of nicotine and cotinine in hair samples collected from the gen- eral population, and compared both the corre- sponding levels and the detection limits with those in previous studies. Finally, initial and run- ning costs of HPLC/UV were compared with other analytical methods. As one of the results, the areas of nicotine and cotinine measured by HPLC/UV with column-switching method were 12.9 and 16.9 times greater, respectively, than those without the column-switching method. The amount of nicotine and cotinine in hair was significantly correlated to number of cigarettes smoked per day (r = 0.228, p = 0.040). In addition, the HPLC/UV method showed similar sensitivity and detection limit (nicotine, 0.10 ng/mg; coti- nine, 0.08 ng/mg) as reported in previous stud- ies. The cost of the HPLC/UV method is lower