Media conditioned by cultured cells represent an excellent source rich in endogenous peptides. Unbiased mass spectrometric analysis of the constituent peptides provides an opportunity to look into proteolytic events such as bioactive peptide processing, membrane protein ectodomain shedding, or even regulated intramembrane proteolysis. If conducted on a large scale, peptidomics has the potential to pinpoint primary cleavage sites. Here a method is described for isolating peptides from cultured cell conditioned media before mass spectrometry analysis.