This work presents a novel method for experimentally quantifying interfacial line tension, which can be readily applied to study a wide variety of different lipid mixtures exhibiting phase coexistence. The method combines AFM imaging of lipid domain nucleation with classical nucleation theories. The results, using symmetric and asymmetric domains, permit the prediction of key physical parameters (critical nuclei size and nucleation rate) in multicomponent bilayer systems with implications toward understanding the dynamic nature of submicrometer domains (i.e., lipid rafts) in cell membranes.