Real-time characterization of digital microfluidic lab-on-a-chip devices is important for biological and chemical applications in which the properties of the microdroplet are time variant. In this paper, a method for in situ characterization of microdroplet interfacial properties is introduced. The proposed characterization method relies on two submodules, namely the contact angle and capacitance sampling submodules, in a digital microfluidic system. In the contact angle measurement submodule, the microdroplet profile is acquired and an accurate contact angle is determined. In the capacitance sampling submodule, the capacitance of the system is measured by means of an activation voltage signal. For verification purposes, the results obtained from the proposed method are compared to the Lippmann–Young equation. The results are in excellent agreement with previously reported values. Finally, the proposed submodules are used to characterize the interfacial properties of a microdroplet containing an aqueous solution of bovine serum albumin (BSA) in which adsorption is a predominant effect. The results show the temporal behaviour of both microdroplet interfacial properties and dielectric characteristics.