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Insulin is a hormone produced by β-cells of the pancreas and controls the amount of sugar in the blood. Since its discovery over 100 years ago, insulin has been used as a life-saving treatment for people with diabetes. Historically, the biological activity or bioidentity of insulin products has been assessed using an in vivo model. However, reduction in animal experiments is a goal for many worldwide, and there is a need to develop in vitro bioassays to reliably test the biological activity of insulin products. This article describes an in vitro cell-based method to assess the biological activity of insulin glargine, insulin aspart, and insulin lispro in a step-by-step manner.