In vitro toxicity tests such as cytotoxicity, mutagenicity and genotoxicity assay are useful for evaluating the relative toxicity of smoke or smoke condensates obtained from different cigarette configurations. A major disadvantage of these tests is relatively time-consuming, complicated and expensive. Recently, a cell-free glutathione consumption assay (GCA) as a rapid and simple screening method for the toxicity assessment of smoke has been reported by Cahours et al. (CORESTA, 2006). This study was carried out to assess the GCA application capable of predicting the toxicity of gas/vapor phase (GVP) of cigarette smoke and to identify individual compounds responsible for the glutathione (GSH) consumption in smoke. Each GVPs from 2R4F, standard cigarette, carbon filter cigarette (ExC) and new carbon filter cigarette (ExN), test cigarettes were collected by automatic smoking machine and evaluated the relative toxicity by GCA and neutral red uptake (NRU) assay. Toxic compounds existed in smoke were also chosen, relative toxicities of these compounds were screened by using two methods and compared individually. The overall order of toxicity by GCA was 2R4F > ExC > ExN, which was consistent with the result of Neutral Red Uptake assay. The levels of carbonyl compounds of ExN were lower than those of 2R4F and ExC, indicating that GSH consumption was associated with carbonyl compound yields. A major toxicant under current study is acrolein, which contributed to more than half of the GSH consumption. Collectively, the toxicity of GVP determined by GCA method may be mainly attributed to acrolein.