Abstract: The bitter compounds in cigarette smoke have non-volatility, diversity and low-abundance. Sensory-oriented separation followed with liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) was proved as a feasible solution for exploring unknown bitter compounds. In this research, water-soluble particulate of cigarette smoke was separated by gel chromatography into several fractions, which were tasted by six experienced reviewers to orient the bitter fractions. Then the bitter fractions were combined to obtain bitter-characteristic component. Sixty-five compounds existing both in this component and in any bitter fraction were preliminarily found with Q-Exactive, a LC-HRMS instrument, using retention time and accurate molecular weight as filter. Sixteen structural formulas of these compounds were confirmed with LC-MS/MS, using retention time and secondary mass spectra of supposed standard substances as references. Finally the proved standard compounds were dissolved in water and tasted by six experienced reviewers. The result shows that ten bitter compounds in bitter-characteristic component were confirmed by more than half of them, which are anatabine, nornicotine, myosmine, cotinine, 2-n-butylimidazole, 2-isopropylimidazole,nicotinamide, N-methylnicotinamide, N-ethylnicotinamide and 3-ethyl-4-methyl-3-pyrrolin-2-one. This method is helpful to identify the key bitter ingredients of cigarette smoke, directionally design cigarette products so as to improve the sensory comfort.