Tobacco stalks are an under-utilized material that remains after the harvest of leaves. Given the share of large leaf tobacco in total production in Serbia (more than 85%) stalks could be easily available as well as payable raw material in the fabrication of cellulose. The aim of this study was to determine cellulose content in leaves and more importantly in stalks of large leaf tobacco and to provide necessary data to processors engaged in processing of biomass. Stalks and leaves of two types of tobacco, Burley and Virginia, reared in Srem, were used as experimental material samples. Two parallel methods were applied for determining cellulose content: method by Kürschner-Hanack and method by Updegraff. It can be concluded that both methods are applicable to tobacco, but the method by Kürschner-Hanack was found to be simpler, more convenient, faster and economically more cost-effective. Thus, it should be recommended for use. Results showed that stalks of large leaf tobacco contain on average 30.50% to 34.30% of cellulose. There was no statistically significant difference in the cellulose content between the two applied methods. Given the number of plants that lag behind in fields after harvesting leaves, 28,000 tons of cellulose could be used.