Peroxisomes are ubiquitous subcellular organelles of eukaryotic cells. As with all organelles, peroxisomes can be purified from cell lysates using a combination of differential centrifugation and density gradient centrifugation. Here, we describe a method for purifying peroxisomes from the yeast Saccharomyces cerevisiae. The method involves gentle lysis of yeast spheroplasts, followed by differential centrifugation to obtain a crude organelle pellet enriched for peroxisomes and mitochondria. To separate peroxisomes from mitochondria, the organelle pellet is resuspended and spun through a sucrose density gradient. Peroxisomes purified in this way can be used to explore whether a protein of interest might be associated with the organelle.