Cellular model serves as a crucial preclinical research tool, providing essential insights into the mechanistic aspects of disease biology. Particularly in the study of chronic metabolic disorders such as type 2 diabetes mellitus and obesity, palmitate (a saturated fatty acid) is often used as a key inducer of insulin resistance in vitro. Within this chapter, I delineate procedures aimed at inducing insulin resistance in AC16 human cardiac-derived cells. The method involves the strategic application of heightened concentrations of palmitate and glucose to specifically target the AKT/GLUT-4 signaling pathway.