Although massively consumed all over the world, cheese, an ancient and much appreciated food, is much more than just a food product. In fact, there are hundreds of different cheese types and many of them reflect the history and cultural identity of a region. Despite being an industrial or traditional food product made using ancient techniques, cheese is a very complex system. Cheese ripening consists of a series of chemical, biochemical and microbiological events that are not only complex but also variable according to the type of cheese being produced. Due to their unique characteristics many cheeses are classified as PDO (Protected Denomination of Origin). These are products of high commercial value subject to legislative and proper labelling regulations and have distinct characteristics associated with several factors such as a geographical area of production, materials and technology used. As a fermentation product, the dynamics of microbial evolution and their metabolic activity is certainly one important factor to take into consideration, since many of the changes occur in cheese during ripening in an interrelated way, in which initial events give rise to products that are subsequently used or modified. Proteolysis is one of the most important biochemical events for the development of flavour and texture during ripening of most cheese varieties and any analytical procedure based on protein analysis must take into consideration that proteolytic activity is a key aspect of cheese manufacture. Similar to other analytical approaches, proteolytic profiles can also be a relevant tool for authenticity studies. Each cheese type can be considered a distinct entity due to the microbiological, proteolytic and lipolytic profiles in the finished product that affect its texture and flavour. In this chapter, the major biochemical processes of cheese ripening will be addressed, focusing particularly on the milk proteins and the role of proteolysis on cheese ripening.