Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption. Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption.Read less <