Poliovirus has been the subject of research for many virologists during the last 80 years. Research on the poliovirus biology has helped to understand the molecular basis of many biological processes, particularly RNA replication and virus–host cell interactions. Laboratory research has also been instrumental in the development of effective and safe vaccines against poliomyelitis and continues to be critical for the assessment of the quality of vaccines used in humans. The work carried out in diagnostic laboratories located in most countries of the world to isolate and characterize poliovirus from clinical samples is also essential to support the Global Polio Eradication Initiative, the most ambitious public health effort ever attempted by man. This chapter describes the most common techniques used in laboratories to isolate, identify, and characterize poliovirus isolates. They include cell culture isolation and typing techniques, virus purification methods, and molecular analyses of viral nucleic acids.