Protein aggregation and amyloid formation are linked to numerous degenerative diseases, such as Alzheimer's or Parkinson's disease. Additionally, protein aggregation plays a crucial role in various biological processes, such as storage of molecules or cell signaling. Protein molecules can form a wide range of aggregates, from oligomers of different sizes to non-specific aggregates and highly ordered cross-β structured amyloid fibrils with diverse morphologies. Circular dichroism (CD) spectroscopy is a widely used technique to study protein structures providing detailed information at the secondary structure level, and is ideal to distinguish and characterize protein aggregates. Despite its potential, CD spectroscopy is often perceived as having limited application on protein aggregates due to challenges, such as sample inhomogeneity, precipitation, light scattering and other factors that complicate accurate analysis. In this study, we present a detailed protocol for examining the structure of protein aggregates and amyloid fibrils using CD spectroscopy. We outline the optimal experimental conditions for sample preparation and demonstrate how to identify and mitigate various interfering effects, using specific examples of disease-related amyloidogenic proteins. We also discuss the instrumental parameters, baseline subtraction, normalization, and quality control of CD spectra. Furthermore, we evaluate the performance of different secondary structure estimating algorithms on amyloid fibril CD spectra highlighting the superiority of BeStSel and CDNN. Our findings could enhance the structural analysis of protein aggregates, contributing to a better understanding of associated diseases and the development of new therapeutic strategies.