Amyloid proteins are considered to play an important role in many human diseases, such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, type II diabetes, and in the prion diseases [1]. These diseases are associated with the formation of inter-and intracellular inclusions that mainly contain insoluble amyloid fibrils, exhibiting a characteristic cross-β secondary structure. These fibrils are∼ 10 nm in diameter and can be several microns in length, and are thus composed of many thousands of the constituent monomeric proteins [2]. However, it still remains unclear why these proteins become cytotoxic upon aggregation. While much research has been done on the monomeric protein and the fibrillar aggregates [1, 3, 4], it is only since the end of the 1990s that attention has shifted from the fibrils to soluble amyloid oligomers as the primary cause of cytotoxicity. Oligomers are aggregation intermediates that precede the formation of fibrils (Figure 6.1). There is growing evidence that suggests that the oligomeric form may play a primary role in the mechanisms of many amyloid diseases [5, 6], with fibrils likely to be inert bystanders in the disease process [7–9]. Cellular toxicity studies show that oligomers have a higher cytotoxicity than the fibrillar form of the proteins [5, 10–14]. Molecular insights into the structure and composition of these oligomeric aggregates are essential for understanding the aggregation process and, ultimately, the cause of the disease. Despite the fact that the oligomeric form is a very important intermediate species, information on the structural and compositional properties is very limited, due to the extremely low concentrations and transient nature of these oligomers. The many reports in the literature present a heterogeneous picture of oligomeric species in terms of size, morphology, toxicity, and method of preparation or purification. The essential underlying question is whether there are structural or compositional similarities among these oligomers and how these similarities can be quantitatively measured, which remains a particular challenge in the field. In this chapter, we will briefly summarize the broad range of biophysical techniques that are available to investigate the structure and composition of amyloid oligomers. We will discuss the relative merits and disadvantages of the