There has been growing interest in the mechanical behaviour of skin due to the rapid development of microneedle devices for drug delivery applications into skin. However, most in vitro experimentation studies that are used to evaluate microneedle performance do not consider the biomechanical properties of skin or that of the subcutaneous layers. In this study, a representative experimental model of skin was developed which was comprised of subcutaneous and muscle mimics. Neonatal porcine skin from the abdominal and back regions was used, with gelatine gels of differing water content (67, 80, 88 and 96%) to represent the subcutaneous tissue, and a type of ballistic gelatine, Perma-Gel®, as a muscle mimic. Dynamic nanoindentation was used to characterize the mechanical properties of each of these layers. A custom-developed impact test rig was used to apply dense polymethylmethacrylate (PMMA) microneedles to the skin models in a controlled and repeatable way with quantification of the insertion force and velocity. Image analysis methods were used to measure penetration depth and area of the breach caused by microneedle penetration following staining and optical imaging. The nanoindentation tests demonstrated that the tissue mimics matched expected values for subcutaneous and muscle tissue, and that the compliance of the subcutaneous mimics increased linearly with water content. The abdominal skin was thinner and less stiff as compared to back skin. The maximum force decreased with gel water content in the abdominal skin but not in the back skin. Overall, larger and deeper perforations were found in the skin models with increasing water content. These data demonstrate the importance of subcutaneous tissue on microneedle performance and the need for representative skin models in microneedle technology development.