Recent experiments revealed the prokaryotic ubiquitin-like protein (PUP) to be a signal for the selective degradation of proteins in Mycobacterium tuberculosis (Mtb). By covalently conjugating the PUP, pupylation functions as a critical post-translational modification (PTM) conserved in actinomycetes. Here, we designed a novel computational tool of GPS-PUP for the prediction of pupylation sites, which was shown to have a promising performance. From small-scale and large-scale studies we collected 238 potentially pupylated substrates for which the exact pupylation sites were still not determined. As an example application, we predicted ∼85% of these proteins with at least one potential pupylation site. Furthermore, through functional analysis, we observed that pupylation can target various substrates so as to regulate a broad array of biological processes, such as the response to stress, sulfate and proton transport, and metabolism. The prediction and analysis results prove to be useful for further experimental investigation. The GPS-PUP 1.0 is freely available at: .