Warfarin, a widely used oral anticoagulant, was synthesized along with its deuterated analogue (deuterated warfarin) to investigate the influence of deuterium substitution on physicochemical and spectral properties. The deuterated derivative was prepared using deuterated benzalacetone as the starting material. Comparative evaluation between warfarin and its deuterated form revealed distinct differences in physicochemical parameters, confirming the successful incorporation of deuterium. Spectroscopic analysis showed a shift in the C–H stretching frequency in the IR spectrum and the absence of C–H proton signals in the ^1H NMR spectrum of deuterated warfarin, both indicative of effective deuteration. These findings suggest structural modification at the molecular level due to isotope substitution. However, further drug metabolism studies employing liver microsomal enzyme CYP3A4 are required to validate the metabolic stability and elucidate the in vivo pharmacokinetic behavior of deuterated warfarin. This study provides a foundation for understanding the potential therapeutic advantages of deuterated analogues in improving drug stability and metabolic profiles.