Soft single photon ionization (SPI)-time-of-flight mass spectrometry (TOFMS) has been applied for the quantitative puff-by-puff-resolved characterization of selected toxic species in cigarette mainstream smoke, namely, nitric oxide (NO), acetaldehyde, butadiene, acetone, isoprene, benzene, toluene, ethyl benzene, and xylene. The 2R4F research cigarette was investigated for whole smoke (unfiltered) and gas phase (filtered). It has been demonstrated that the existing smoking regime for total smoke analysis (smoke from a complete cigarette) features deficiencies when applied to puff-resolved measurements. This is especially the case for analysis in which a glass fiber filter is used to separate the smoke particulate and gas phases because material is desorbed from the filter and influences succeeding puffs. Regarding whole smoke measurements, succeeding puffs are affected by contamination and memory effects of the smoking machine itself, but to a lower extent. Quantitative puff-resolved smoke profiles show that the puff yields of the various constituents can differ tremendously. Most species' concentrations increase gradually with puff number, which is mainly due to the reduction in length of the cigarette. However, high yields in the first puff are observed for butadiene and isoprene, suggesting different formation mechanisms for these compounds. First results are presented in which these high yields in the first puff are mainly associated with the gaseous fraction of the smoke.