Peroxisomes are ubiquitous organelles with essential roles in human health, and their dysfunction leads to a spectrum of genetic disorders. Visualization of peroxisomes is key to identifying peroxisome dysfunction; however, progress has been limited by the lack of peroxisome-specific probes. By developing PeroxiSPY, peroxisome-specific probes that mimic natural peroxisome metabolites, we enabled real-time detection, tracking, and quantification of peroxisome dynamics in live mammalian cells. Additionally, the applications of this technique to patient cells with peroxisome disorders reveal pathological phenotypes. Here, we provide a detailed protocol for the detection of peroxisomes in live mammalian cells, as well as the detection of peroxisomes post-staining in fixed cells. We demonstrate how to stain peroxisomes in live cells using peroxisome-specific probes, focusing on the preparation of cells, timing of the procedure, reproducibility, and troubleshooting. These probes enable rapid, specific, and non-toxic live-cell detection of peroxisomes, offering a unique, quantitative method for assessing peroxisome function and its dysfunction in peroxisome-related disorders.