Background: According to the American Diabetes Association (ADA), individuals with diabetes should limit their milk consumption to no more than 1 cup a day. This is due to the presence of lactose in milk, a sugar that can increase blood glucose levels. However, this restriction can affect the quality of life of milk lovers who may miss out on milk’s nutritional benefits. To solve this problem, many milk substitutes have emerged in the market, such as almond, rice, and soy milk. Most provide 75% less protein than cow’s milk, while soy milk (12.5% less protein) is controversial. A recent emerging technology, cell-free protein synthesis (CFPS/TXTL), may allow the synthesis of a series of enzymes needed to make dairy more diabetic-friendly. The enzyme synthesis method has the potential to decrease production costs due to higher yield and easier purification. Purpose: The main aim of this project is to convert milk lactose into allulose with enzymes produced using CFPS/TXTL, thus providing a better milk option for individuals with diabetes. Milk with allulose, a rare sugar, may be superior to lactose-free milk because it is less sweet, has fewer calories, does not cause blood glucose spikes, and may be considered a functional food. Methods: The first step focuses on converting lactose into glucose and galactose using lactase enzyme. This enzyme was first synthesized in the lab using a plasmid containing the gene for lactase (LacZ) as template for TXTL. Following an 18-hour incubation, the lactase enzyme produced was quantified using Qubit™ 4 Fluorometer. The efficiency of the cell-free generated lactase was then compared against commercially prepared powdered lactase using an ONPG-inspired protocol. After the conversion of lactose into glucose and galactose was achieved, the prospect of implementing an additional enzymatic cascade to further convert these substrates into allulose was considered. However, due to the temporal limitations inherent in this study, the exploration of the supplementary enzymatic cascade was not attempted. Results: The Qubit fluorometer recorded concentrations of 3.66 mg/mL, 4.75 mg/mL, and 3.62 mg/mL in the experimental samples, suggesting the presence of protein in the form of lactase enzyme. The ONPG-based test showed an enzyme that is equally if not more effective than its commercially manufactured equivalent. Discussion: The TXTL or Cell-Free Protein Synthesis (CFPS) method yielded high quantities of the lactase enzyme which has in some instances shown to have equal if not higher efficiency than commercial powdered lactase. Conclusion: Knowing that the production of lactase using CFPS hasn’t been attempted before, the results are promising. The emerging cell-free protein synthesis technology has succeeded in generating a substantial yield of an enzyme, sometime surpassing its commercially produced equivalent. However, further research is needed to achieve the comprehensive complete transformation of lactose into the intended final product, allulose.