Anisotropy exists in many soft biological tissues. The most common anisotropy is transverse isotropy, which is typical for fiber-reinforced structures, such as the brain white matter, tendon and muscle. Although many methods have been proposed to determine tissue properties, techniques to characterize transversely isotropic materials remain limited. The goal of this study is to investigate the feasibility of asymmetric indentation coupled with numerical optimization based on inverse finite element (FE) simulation to characterize transversely isotropic soft biological tissues. The proposed approach combining indentation and optimization may provide a useful general framework to characterize a variety of fiber-reinforced soft tissues in the future.