DNA double-strand breaks (DSBs) are the most harmful lesions to DNA in the cell. To cope with these insults, all organisms have devised two main types of evolutionary conserved mechanisms for their repair, homologous recombination (HR), and non-homologous end joining (NHEJ). The first one operates predominantly during the S/G2 phase of the cell cycle, when the sister chromatid is available for repair. Because HR requires homology for repair, this pathway is considered essentially error-free. NHEJ is the pathway of choice in the other phases of the cell cycle, including G1. This pathway is generally faithful but can be prone to errors. Most spontaneous DNA breaks arising in somatic cells occur randomly as a consequence of DNA replication failure caused by either DNA lesions or generated by obstacles that impede the progression of the replication fork (e.g., protein-bound to DNA, DNA secondary structures, replication–transcription conflicts, etc.). For this reason, HR is a major DNA repair pathway during S/G2 phases of the cell cycle. Thus, HR is intimately ligated to the prevention of genome instability in replicating somatic cells. In meiotic cells however, DSBs are developmentally controlled by the action of specific endonucleases where HR is essential; gametogenesis is not possible in the absence of HR. Genome instability and in particular defective HR is a common feature of a number of genetic diseases including cancer. Defects in HR in meiotic cells can lead to birth defects such as Down syndrome. Considering the relevance of HR as one of the major DSB repair pathways in mitotically cycling cells, as well as its essential role in meiosis, understanding the molecular mechanisms and factors that participate in HR is of key importance in Molecular Biology and Biomedicine. In this book, we compile a series of laboratory protocols covering the analysis of different steps of the homologous recombination process from the genetic, molecular biology, and cell biology perspectives. As these steps are very well conserved through evolution, taking advantage of different model organisms have led to accelerated discoveries in this field. Thus, when appropriate, some of the protocols we present here are explained in the context of more than one model system. We hope this book will facilitate the use of both classical and more recent approaches to answer specific questions on HR mechanisms as well as to decipher the function of novel factors involved in HR. We expect that this compilation of protocols elaborated by leading experts in the field will be useful not only to the scientific community working in genome integrity but also to scientists working in other areas such as cancer biology or cell cycle with renovated interests in HR and DSB repair.