Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is not a harmful substance except for the addiction it causes. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of NNN, NNK, and NNAL along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The LOD and LOQ were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.365 mg/kg, 0.344 mg/kg, and 5.71 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.062 mg/kg, 0.036 mg/kg and 0.97 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect’s whole body and faeces. Recoveries ranged between 95-98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds.