Proteomics provides a powerful tool to characterize the protein content of an organelle. However, identifications obtained through mass spectrometry and database searching only make sense if the organelle sample is not heavily cross-contaminated. Besides the proteomic analysis, which gives an overview of possible cross-contamination, biochemical methods can be used to assess sample purity. These methods use specific markers that are detected and measured. Here, we describe the use of immunological, enzymatic, lipid, and pigment markers that allow the purity of chloroplast envelope fractions to be estimated.