Research on prion protein (PrP) and pathogenic prion has been very intensive because of its importance as model system for neurodegenerative diseases. One important aspect of this research has been the application of chemical biology tools. In this review we describe new developments like native chemical ligation (NCL) and expressed protein ligation (EPL) for the synthesis and semisynthesis of proteins in general and PrP in particular. These techniques allow the synthesis of designed tailor made analogs which can be used in conjunction with modern biophysical methods like fluorescence spectroscopy, solid state Nuclear Magnetic Resonance (ssNMR), and Electron Paramagnetic Resonance (EPR). Another aspect of prion research is concerned with the interaction of PrP with small organic molecules and metals. The results are critically reviewed and put into perspective of their implication for PrP function.