Protein misfolding, which literally means “incorrect folding” is associated with a number of pathological states in humans, collectively termed protein misfolding diseases. In some cases the disease arises because a specific protein is no longer functional when adopting a misfolded state or undergoes a severe trafficking impairment (loss of function diseases). In most diseases, however, the pathological state originates because misfolding occurs concomitantly with aggregation and the underlying aggregates are detrimental (gain of function diseases). The question as to why proteins misfold and self-assemble into well-organised fibrillar aggregates characterised by an extensive β-sheet structure, generally referred to as amyloid or amyloid-like fibrils, is very complex and cannot be answered exhaustively even after several pages of a book. In spite of these difficulties it is fascinating to try to address this issue, particularly by describing a few concepts that are well established, have general validity, and benefit from a general consensus in the scientific community. In this book chapter we described our current knowledge on why and how proteins misfold in vitro. Considerable attention was paid to the conformational changes that represent the starting point of misfolding phenomena and to the sequence determinants that enable the resulting misfolded, yet conformational states to self-assemble into ordered fibrillar aggregates. Finally, we mentioned all the factors that are thought to influence protein misfolding in an in vivo context.