This article summarizes the most important developments in the use of 2D-LC for bioanalysis in the last 5 years. While several interesting and powerful applications have been developed recently, this work has been supported by continued, significant development of theoretical concepts, instrument development and practical aspects of method development. Some of the most exciting applications have been focused on the use of 2D-LC and characterize proteins both as biotherapeutic drug substances, and in formulations. These materials are inherently complex, difficult to resolve chromatographically and present problems that are essentially unknown (e.g., aggregation) in the small molecule world, thus 2D-LC can be leveraged very effectively to address these challenges.