Previously, we developed a biochemical reaction simulator called WinBEST-KIT (Biochemical Engineering System analyzing Tool-KIT, which runs under Microsoft Windows) for analyzing complicated metabolic pathways. WinBEST-KIT provides an integrated simulation environment for experimental researchers in metabolic engineering. A particularly notable feature of WinBEST-KIT is that users can easily define and customize reaction symbols in the graphical user interface. Users can use their original kinetic equations, in addition to the pre-installed standard kinetic equations, to represent unknown kinetic mechanisms as reaction steps. However, owing to the increasing size of reaction systems to be analyzed in metabolic pathways, large-scale reaction systems must be divided into several arbitrary compartmental reaction systems and procedures are needed, such as multilayered hierarchical representation, to describe the interactions between the compartmental reaction systems. Accordingly, in this study, we developed a new version of WinBEST-KIT that enables users to construct several arbitrary reaction schemes as layers, to connect the layers, and to analyze the interactions between them. This hierarchical representation is effective for constructing multilayered mathematical models of biochemical systems, such as genome-enzyme-metabolite systems, reaction cascade systems, and multicellular systems.