Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.