The use of animal testing in the cosmetic industry is already prohibited in more than 40 countries, including those of the EU. The pressure for it to be banned worldwide in the future is increasing, so the need for animal alternatives is of great interest today. In addition, using animals and humans in scientific research is ethically reprehensible. This study aimed to prove some of the anti-aging properties of elastin (EL), hydrolyzed collagen (HC), and two vegan collagen-like products (Veg Col) in a tri-layered chitosan membrane that was ionically crosslinked with sodium tripolyphosphate (TPP). In the first approach, as a way of representing different layers of a biological system, such as the epidermis and the two dermis sublayers, EL, HC, or Veg Col were independently introduced into the two inner layers (2L(i+b)). Their effects were compared with those of their introduction into three layers (3L). Different experiments were performed on the membrane to test its elasticity, hydration, moisture retention, and pore reduction at different concentrations of EL, HC, and Veg Col, and the results were normalized vs. a blank membrane. This new alternative to animal or human testing can be suitable for proving certain efficacy claims for active ingredients or products in the pharmaceutical, nutritional, and cosmetic fields.