Interactions between proteins control molecular functions such as signalling or metabolic activity. Assembly of proteins via scaffold proteins or in higher-order complexes is a key regulatory mechanism. Understanding and functionally applying this concept requires the construction, study, and utilization of synthetic scaffolds. This chapter first describes protein scaffolding in the context of its natural function as well as the underlying mechanistic origins via mathematical models and simulations. This is then funnelled into examples of synthetic biology approaches to engineer new scaffolds and their usage as regulators of signalling networks and metabolic engineering.