Medication administration remains the hallmark of medication therapy. A general goal of pharmacotherapy is to ensure a therapeutic drug concentration in specific areas of the body or throughout the body. Among the various routes of administration, Transdermal delivery represents an attractive alternative to oral delivery of drugs and is poised to provide an alternative to hypodermic injection too. It provides an easy, reliable mechanism of administering drugs, and bypasses the hepatic first-pass metabolism, providing a more reliable clinical action. However, a major challenge is the barrier function of the skin. Drug transport into and through the skin is still difficult, despite the fact that drugs are necessary in several skin compartments such as the epidermis, dermis, and hair follicles to effectively treat skin diseases. To increase drug penetration and permeation, a better understanding of skin barrier physiology is required. We summarize our current knowledge in this book about mechanical barriers, including skin, and then critically compare the applicability of several physical, biochemical, and microscopic methods to measure the permeation of drugs through these barriers.