A method for the simultaneous determination of volatile and semi-volatile fatty acids in TPM of mainstream cigarette smoke was developed. The organic acids in TPM of mainstream cigarette smoke were extracted with dichloromethane in an ultrasonic bath, then were derivertised with N,O-Bis(trimethylsilyl)trifluoroacetamide. The derivatives of 20 organic acids were analyzed by selective ion monitoring GC/MS with the recoveries at the range of 74.3% to 101.8%, and R.S.D in overall analysis procedure was less than 9.8% for all organic acids. Applying this method, the puff-by-puff deliveries of the 20 volatile and semi-volatile organic acids in TPM of mainstream cigarette smoke were investigated. Puff-by-puff smoking was performed on a modified smoking machine. The TPM of individual puff of 20 cigarettes was collected on a Cambridge filter according to the ISO method. The yields of 20 organic acids in TPM of the seven puffs were determined quantitatively. The results indicated that there is an ascending trend that is similar to TPM in which the organic acids delivery increases with increasing puff count. However, the puff-by-puff delivery profile of volatile and semi-volatile organic acids is dissimilar.