Systematic dissection of the ubiquitylation proteome is emerging as an appealing but challenging research topic because of the significant roles ubiquitylation play not only in protein degradation but also in many other cellular functions. High-throughput experimental studies using mass spectrometry have identified many ubiquitylation sites, primarily from eukaryotes. However, the vast majority of ubiquitylation sites remain undiscovered, even in well-studied systems. Because mass spectrometry-based experimental approaches for identifying ubiquitylation events are costly, time-consuming and biased toward abundant proteins and proteotypic peptides, in silico prediction of ubiquitylation sites is a potentially useful alternative strategy for whole proteome annotation. Because of various limitations, current ubiquitylation site prediction tools were not well designed to comprehensively assess proteomes.