Many viruses with compact RNA genomes use a replication strategy involving the translation and proteolytic processing of a polyprotein. Astroviruses include a range of human and animal species and can infect cells of intestinal and neuronal origin. These viruses have positive-sense RNA genomes and translate their nonstructural polyprotein, which is subsequently processed by viral and cellular proteases. The polyprotein cleavage sites are predicted but not experimentally confirmed. Using N-terminomics and a complementary range of other experimental techniques, we mapped polyprotein cleavage sites in classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2, providing a map of individual proteins and their boundaries. Furthermore, we characterize processing precursors and replication properties of processing-defective mutant replicons, further elucidating the importance of polyprotein cleavage in astroviruses. Understanding polyprotein processing dynamics is essential for interpreting viral infection stages and identifying new drug targets and antiviral strategies.