Harmful and potentially harmful constituents (HPHCs) are chemical compounds in tobacco smoke that cause harm to smokers and non-smokers. This study established and validated methods for the analysis of HPHCs from mainstream cigarette smoke. The analyzed HPHCs were categorized into seven groups: aromatic amines, volatile organic compounds (VOCs), heavy metals, tobacco specific nitrosamines (TSNAs), benzo[a] pyrene (B[a]P), ammonia, and carbonyl compounds. The methods were validated by specificity, linearity, limit of detection (LOD), accuracy, precision, and recovery. These validated methods were then applied to the reference cigarettes (1R5F, 3R4F). The correlation coefficients (r(2)) for the calibration curves of the seven groups were over 0.995. The LODs showed values of 0.01-0.04 ng/cig cig for aromatic amines, 0.01-0.16 mu g/cig for VOCs, 0.01-1.27 ng/cig for heavy metals, 0.06-0.28 ng/cig for TSNAs, 0.04 ng/cig for benzo[a] pyrene, 0.08 mu g/cig for ammonia, and 0.78-1.77 mu g/cig for carbonyl compounds. The precisions obtained from the intra and inter-day batches were less than 15%. The accuracy and the recovery range were less than 15% and 79.2-117.5%, respectively. The proposed methods can therefore be applied for determining HPHCs in tobacco mainstream smoke.