Abstract: In order to investigate the differences in puff-by-puff release of alkaloids in mainstream smoke between slim and normal cigarettes, a method for simultaneous determination of five alkaloids (nicotine, nornicotine, myosmine, anabasine and anatabine) on a puff-by-puff basis was developed, and eight cigarette samples were analyzed with the developed method. The results showed that:1) The calibration curves presented a good linearity for the five alkaloids with the correlation coefficients of 0.999 0-0.999 6. Recoveries at different spiked levels ranged from 80.41% to 107.09% and the intra-and inter-day relative standard deviations (RSDs) were from 2.46% to 9.70%. 2) Generally, the puff-by-puff releases of TPM, nicotine, nornicotine, myosmine, anabasine and anatabine in mainstream smoke of slim cigarettes were lower than those of normal cigarettes. Non-parametric test indicated that there were significant differences in puff-by-puff releases of TPM, nicotine, nornicotine, anabasine and anatabine in mainstream smoke between slim and normal cigarettes. However, the release of myosmine did not significantly differ between the two types of cigarettes. This method features good repeatability and accuracy, it is suitable for the puff-by-puff analysis of the five alkaloids in mainstream cigarette smoke.