Synthetic agents that disrupt intracellular protein protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, fusicoccin, which binds 14-3-3 protein with submicromolar affinity. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. compound 3, but not 1 or 2, disrupts endogenous 14-3,3/cRaf intetactions, suggesting that cell death is caused by inhibition a 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs.