Positive-sense RNA viruses often encode large polyproteins that are proteolytically processed by viral and host proteases into functional replication proteins. Astroviruses infect intestinal and neuronal cells across diverse human and animal hosts and also rely on polyprotein cleavage for replication. In this study, we mapped the cleavage sites of the nonstructural polyproteins of classical human astrovirus 1 (HAstV1) and neurotropic astrovirus strain MLB2 using complementary N-terminomics of in