People with a lower limb amputation have altered motion strategies during daily tasks compared to people without an amputation. These altered motion strategies can result in the development of secondary conditions, such as residual limb or low back pain, but the underlying muscle activity that causes altered motion strategies, and potentially pain development, is often unclear. For people with a lower limb amputation, prosthetic alignment is an important component that may contribute to altered motion strategies and can be easily adjusted in a clinical setting. However, the effect of altered prosthetic alignment on muscle activity is not well understood, especially during sit-to-stand, which is an important activity of daily living. Therefore, the purpose of this work was to evaluate the effect of altered prosthetic alignment on muscle activity for people with a unilateral transtibial amputation (TTA) during sit-to-stand. Characterizing this muscle activity will contribute to the understanding of altered motion strategies associated with TTA and how they may contribute secondary conditions. Further, this work will increase our knowledge of the effects of prosthetic alignment, which is important for improving alignment prescription guidelines.