Regular insulin can reduce hyperglycemia when directly added to total parenteral nutrition (TPN) solutions. Insulin is not routinely added to all TPN solutions. For patients who require insulin prior to the initiation of TPN supplement, one-third to one-half of the usual total daily dose can be added to the TPN bag as regular human insulin. However, an incorrect dose or an interaction between insulin and the TPN bag material may affect blood sugar control in clinical practice. Therefore, it is important to quantitatively determine the final dose of insulin in the TPN bag. High performance liquid chromatography is a very powerful technique for determining the purity of proteins. The goal of this study was to use high-performance liquid chromatography to perform quantitative analysis of insulin in a TPN bag. The analysis was performed under different light conditions (UV, fluorescent, and darkness) and different temperatures (25°C and 2–8°C). The results show that adsorption of insulin on an ethylene vinyl acetate TPN bag is significantly higher than that on glass. Based on the results, it is evident that regular insulin should be administered separately from TPN to reduce cost and eliminate wasteful disposal of TPN solutions.