Abstract Recent work has made it clear that pericentriolar material (PCM), the matrix of proteins surrounding centrioles, contributes to most functions of centrosomes. Given the occurrence of centrosome amplification in most solid tumors and the unconventional survival of these tumor cells, it is tempting to hypothesize that gel-like mitotic PCM would cluster extra centrosomes to defend against mitotic errors and increase tumor cell survival. However, because PCM lacks an encompassing membrane, is highly dynamic, and is physically connected to centrioles, few methods can decode the components of this microscale matrix. In this study, we took advantage of differential labeling between two sets of APEX2-centrosome reactions to design a strategy for acquiring the PCM proteome in living undisturbed cells without synchronization treatment, which identified 392 PCM proteins. Localization of ubiquitination promotion proteins away from PCM was a predominant mechanism to maintain the large size of PCM for centrosome clustering during mitosis in cancer cells. Depletion of PCM gene kinesin family member 20A (KIF20A) caused centrosome clustering failure and apoptosis in cancer cells in vitro and in vivo. Thus, our study suggests a strategy for targeting a wide range of tumors exhibiting centrosome amplification and provides a proteomic resource for future mining of PCM proteins. Significance: This study identifies the proteome of pericentriolar material and reveals therapeutic vulnerabilities in tumors bearing centrosome amplification.