The isolation of cell organelles from model organisms in high purity is important for biochemical analyses of single proteins, entire metabolic pathways, and protein complexes and is absolutely essential for organelle proteome analyses. The efficient enrichment of nearly all cell organelles is more difficult from Arabidopsis as compared to traditional model plants and especially challenging for peroxisomes. Leaf peroxisomes are generally very instable in aqueous solution due to the presence of a single membrane and (para-)crystalline inclusions in the matrix. Leaf peroxisomes from Arabidopsis are particularly fragile and, moreover, strongly physically adhere to chloroplasts and mitochondria for largely unknown reasons. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes by Percoll followed by sucrose density gradient centrifugation that yields high purity suitable for proteome analyses. Diverse enzymatic and immuno-biochemical methods are summarized to assess purity and intactness.