Peroxisomes are essential organelles for human health that are present in virtually all nucleated cells. Important functions of peroxisomes include lipid metabolism, the production and breakdown of hydrogen peroxide (H2O2), and antiviral signaling. To preserve cellular homeostasis, dysfunctional and superfluous peroxisomes need to be selectively removed. However, how this process is regulated is currently poorly understood. The major aim of this work was to gain a better insight into the molecular mechanisms governing peroxisome degradation in mammalian cells. Initially, we investigated whether or not oxidatively damaged peroxisomes are targeted for selective removal. For this purpose, we employed the photosensitizer KillerRed to generate highly reactive superoxide radicals within the peroxisomal matrix. Although we were unable to observe peroxisome degradation upon photoactivation of KillerRed, we instead discovered that this protein emits weak green fluorescence upon light excitation at 480 nm, an important finding for researchers working with this increasingly popular photosensitizer. Next, we developed a novel D-amino oxidase-based approach to locally induce peroxisomal H2O2 stress inside the organelles. Using this method, we showed that also excessive intraperoxisomal H2O2-production does not trigger the degradation of peroxisomes. However, by employing the same method, we could demonstrate that peroxisome-derived H2O2 directly and rapidly oxidizes redox-sensitive proteins in the cytosol, and that H2O2-signaling from peroxisomes to mitochondria does not occur via direct diffusion of this reactive molecule between these organelles. As such, we believe that this system is a suitable tool for further studies regarding peroxisomal H2O2-signaling in mammalian cells. Finally, we discovered that PEX5 proteins fused to a bulky C-terminal tag can trigger peroxisome degradation in mouse embryonic fibroblasts. Specifically, we observed that expression of these proteins resulted in an accumulation of Cys11-monoubiquitinated PEX5 at the outside of the peroxisomal membrane, in turn leading to selective peroxisome removal via the autophagy pathway. We believe that this phenotype mimics a physiological situation where PEX5 cargo proteins are unable to be released into the peroxisomal lumen. In summary, this study provides strong evidence that monoubiquitinated PEX5 can serve as a quality control mechanism to eliminate dysfunctional peroxisomes. In addition, it paves the way for future investigations aimed at elucidating the molecular basis underlying selective peroxisome degradation, an essential prerequisite to understand how defects in this process may be linked to clinically-relevant disease phenotypes.