Tobacco　metabolites　play　a　crucial　role　in　shaping　the　sensory　experience　of　tobacco　products,　such　as　cigarettes.　Bitter　metabolites　in　tobacco　not　only　serve　as　protective　agents　in　plants,　but　also　significantly　influence　the　overall　flavor　profile　of　cigarettes.　In　this　study,　we　employed　an　integrated　approach　combining　chemical　profiling,　cell-based　calcium　imaging　assays,　and　molecular　docking　studies　to　identify　key　bitter　compounds　in　cigarette　smoke　and　explore　the　molecular　mechanisms　underlying　their　bitterness.　We　analyzed　22　commercial　cigarette　brands,　revealing　notable　variability　in　bitterness　perception.　GC-MS　analysis　identified　88　compounds　in　mainstream　cigarette　smoke　(MCS),　with　pyridines,　esters,　and　phenols　being　the　most　prevalent.　Using　a　TAS2R14-G　alpha　16gust44-HEK293T　cell-based　assay,　we　objectively　measured　bitterness　and　identified　nicotine,　succinimide,　and　scopoletin　as　key　bitter　compounds　through　correlation　analysis.　Molecular　docking　studies　provided　detailed　insights　into　the　interactions　of　these　compounds　with　the　TAS2R14　receptor,　highlighting　significant　hydrogen　bonding　and　hydrophobic　interactions.　This　integrated　methodology　not　only　identifies　key　contributors　to　cigarette　bitterness　but　also　enhances　our　understanding　of　the　molecular　basis　of　bitterness　perception　in　tobacco.　These　findings　pave　the　way　for　future　studies　to　further　elucidate　the　complex　interactions　of　bitter　compounds.　This　understanding　of　the　links　between　tobacco　bitterness　and　consumption　holds　significant　implications　for　public　health　and　tobacco　control　initiatives.