The gradual transition from egg tempera to oil as the predominant paint binding medium in fifteenth-century Italy is examined in relation to the chemistry of the binders. The impact of the choice of binder on the physical and optical properties of the paint is discussed, together with the effect this has on painting style. In order to identify the paint binder, instrumental methods of analysis are used to detect characteristic chemical components in paint samples. The level of sophistication of these analytical methods has improved significantly over the last couple of decades and the most widespread analytical methods are discussed, together with more recent developments. This analytical approach is shown to have significantly enhanced our understanding of the transition from egg tempera to oil-based techniques that took place in Italy in the fifteenth century.