Layer-by-layer (LbL) assembly is the process of building functional multilayered thin films. Owing to its highly modular and versatile nature, it has been used to coat a wide variety of different surfaces, including inorganic substrates, membranes, implants, nanoparticles and even living cells. It provides ways to induce responsiveness through both the chemically engineered macromolecular components, and the way the multilayers are built up. For example, assembly degradability can be adjusted by using degradable polymers or crosslinkers, while physical properties can be altered through the use of additives, or by the assembly method. This chapter is dedicated to LbL fabrication-specific responsiveness, and to recent developments in multilayers composed of specifically tailored polymers. It further focuses on chemically and biologically responsive LbL systems, with main applications in the biomedical field. The introduction covers general aspects of LbL assembly and physicochemical aspects of the assemblies. The second part describes physicochemical aspects in more detail with examples on how variation in deposition conditions, e.g. pH and ionic strength, as well as specific additives, induce responsiveness to the resulting multilayers. It also highlights several reports on compartmentalized multilayered coating fabrication for tunable disassembly or release of incorporated materials. The third part describes multilayers fabricated with chemically tailored biomaterials for different chemical and biological responsiveness. More specifically, multilayer disassembly can be triggered by the inherent responsiveness of one of the multilayer components, through incorporation of labile bonds that respond to specific external triggers, or through disruption of the interlayer interaction between two or more multilayer components.