The human body has several barriers that protect its integrity and shield it from mechanical, chemical, and microbial harm. The various barriers include the skin, intestinal and respiratory epithelia, blood–brain barrier (BBB), and immune system. In the present review, the focus is on the physical barriers that are formed by cell layers. The barrier function is influenced by the molecular microenvironment of the cells forming the barriers. The integrity of the barrier cell layers is maintained by the intricate balance of protein expression that is partly regulated by microRNAs (miRNAs) both in the intracellular space and the extracellular microenvironment. The detection of changes in miRNA patterns has become a major focus of diagnostic, prognostic, and disease progression, as well as therapy-response, markers using a great variety of detection systems in recent years. In the present review, we highlight the importance of liquid biopsies in assessing barrier integrity and challenges in differential miRNA detection.