<p>Cell-based foods are anticipated to be a sustainable alternative to animal protein sources. The primary challenge is developing a food-grade serum-free medium (SFM) that is affordable, safe, acceptable to consumers, and available in large quantities. This study was conducted to develop such a medium using food ingredients dissolved in water that support the expansion of bovine myoblasts and maintain their differentiation capability. Through screening of various food ingredients, whey was identified as the most effective component. Whey improved myoblast proliferation in SFM to levels than those in a serum-containing medium. The differentiation capability of myoblasts cultured in whey-containing SFM was similar to that in serum-containing medium. Remarkably, whey could support myoblast proliferation even after heat treatment, allowing for the sterilization of whey-containing media by heat treatment, a common process in food processing. Hence, whey could be an ideal ingredient for affordable food-grade serum replacement in cell-based food production.</p>