Post-translational modification of p53 by ubiquitin resides in the center of a fine-tuned regulatory network that activates the tumor suppressor in response to genotoxic stress. Inhibition of p53 ubiquitination by DNA damage not only prevents p53 from degradation but also promotes its nuclear accumulation leading to transactivation of a number of downstream genes that are essential for the control of cell cycle progression, cell survival, and cellular senescence. Therefore, there are growing interests in studying p53 ubiquitination under physiological/pathological conditions. We describe herein a cell-based method and an in vitro reconstituted assay that are convenient, reproducible, and adaptable for various experimental conditions for detection of p53 ubiquitination. Wide application of these methods in studying mechanisms underlying regulation of p53 ubiquitination shall assist us in better understanding of the function of the tumor suppressor.