Abstract: Protein post-translational modifications (PTMs) are involved in essentially all aspects of cellular signaling. Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential PTMs in eukaryotes. Unfortunately, probing the function of these modifications in live cells, is a challenging task. While chemical protein synthesis (CPS) offers great opportunities in studying Ub and SUMO biology, the low cell permeability of proteins limits these studies mainly for in vitro applications. Therefore, delivering synthetic proteins designed to answer specific biological questions into live cells, is a promising approach with applications for basic research and therapeutic developments. Here, we utilize cell-penetrating peptides (CPPs) and physical approaches to stretch the limits of synthetic protein delivery. For this goal, we developed a new palladium promoted chemical activation strategy to inhibit deubiquitinating enzymes in prostate cancer cells. Moreover, we report a new approach to boost CPP mediated protein delivery and for the multiplexed loading of up to four differentially labeled proteins with organic fluorophores. We applied these strategies to probe ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags (e.g., GFP) or perturbation to their endogenous levels. Using external chemical stimuli, we induced in-situ protein activation and various stress conditions to study our synthetic proteins in their native environment in disease models. Our studies reveal unprecedented involvements of Ub and SUMO2 in lysosomes, depending on their conjugation states and provide first evidence of how Ub phosphorylation effects its function during mitophagy. We envision that these approaches will improve our understanding of dynamic PTM dependent cellular processes such as formation and disassembly of membraneless organelles by liquid-liquid phase separation.