A sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for the quantification of Nicotine (NT) and Cotinine (CN) using Continine-d A linear response function was established for the range of concentrations 3-200 (r > 0.995) for NT and 3-600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are stable in the battery of stability studies viz., stock solution, bench-top and auto-sampler. This method was successfully utilized to validate a newly developed preclinical smoking model in mice.