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Summary: We describe the procedures to perform the following: (1) the de novo discovery of mutational signatures from raw sequencing data of viral samples and (2) the association of existing viral mutational signatures to the samples of a given dataset. The goal is to identify and characterize the nucleotide substitution patterns related to the mutational processes that underlie the origination of variants in viral genomes. The VirMutSig protocol is available at this link: https://github.com/BIMIB-DISCo/VirMutSig.For complete information on the theoretical aspects of this protocol, please refer to Graudenzi et al. (2021).