<p>Sterols are essential for the growth, development, and survival of insects. Changes in the dietary sterol composition can cause significant metabolic and physiological effects. In this study, using a sterol-free artificial diet formulation supplemented with one of six sterols including (+)-4-cholesten-3-one, brassicasterol, campesterol, <i>β</i>-sitosterol, cholesterol, and stigmasterol, we investigated the effect of dietary sterols in larval growth of the tobacco cutworm <i>Spodoptera litura</i> (Lepidoptera: Noctuidae). We first confirmed by gas chromatography–mass spectrometry (GC–MS) analysis that our artificial diet contained extremely low amount of sterols. Tobacco cutworm fed the artificial diet supplemented with brassicasterol, campesterol, cholesterol, <i>β</i>-sitosterol, or stigmasterol showed higher survival rate and growth of <i>S. litura</i> larvae compared with those supplemented with (+)-4-cholesten-3-one or without sterol supplementation, leading to the preparation of more optimized diets for <i>S. litura</i> larvae. GC–MS analysis also revealed that the larvae fed on the diet supplemented with (+)-4-cholesten-3-one and without sterols showed little sterol content. This study indicated that sterols whose backbone is the same as that of cholesterol were utilizable by <i>S. litura</i>, while (+)-4-cholesten-3-one, which had different backbone, was not. By utilizing this sterol-free artificial diet, further research will be able to identify utilizable and non-utilizable sterols, establishing a comprehensive framework for sterol utilization in <i>S. litura</i>.</p>