Ubiquitination is an essential protein posttranslational modification in eukaryotes governing protein homeostasis and ubiquitin-mediated cellular signaling. In human cells, the dynamics and substrate-specificity of ubiquitination are mediated by over 600 genetically encoded E3 ligases. Dysregulation of E3 ligase activities could disrupt protein turnover, gene expression, and signal transduction, which is widely implicated in diseases such as cancer and aging-related diseases. In this report, we presented a computational workflow, UbE3-APA, to profile E3 ligase activities based on quantitative proteomics data and bioinformatics analysis. We validated the strategy with published datasets from the analysis of cells and tissues with genetic knockout or knockdown of specific E3 ligases and demonstrated that the strategy can be applied to both data-dependent or data-independent quantitative proteomics datasets.