Disruption of tissue barriers formed by cells is an integral part of the pathophysiology of many diseases. Therefore, a thorough understanding of tissue barrier function is essential when studying the causes and mechanisms of disease as well as when developing novel treatments. In vitro methods play an integral role in understanding tissue barrier function, and several techniques have been developed in order to evaluate barrier integrity of cultured cell layers, from microscopy imaging of cell-cell adhesion proteins to measuring ionic currents, to flux of water or transport of molecules across cellular barriers. Unfortunately, many of the current in vitro methods suffer from not fully recapitulating the microenvironment of tissues and organs. Recently, organ-on-chip devices have emerged to overcome this challenge. Organs-on-chips are microfluidic cell culture devices with continuously perfused microchannels inhabited by living cells. Freedom of changing the design of device architecture offers the opportunity of recapitulating the in vivo physiological environment while measuring barrier function. Assessment of barriers in organs-on-chips can be challenging as they may require dedicated setups and have smaller volumes that are more sensitive to environmental conditions. But they do provide the option of continuous, non-invasive sensing of barrier quality, which enables better investigation of important aspects of pathophysiology, biological processes, and development of therapies that target barrier tissues. Here, we discuss several techniques to assess barrier function of tissues in organs-on-chips, highlighting advantages and technical challenges.