Factor I (FI) is a soluble, 88 kDa glycoprotein present in plasma at a concentration of approximately 35 mg/L. FI inhibits all complement pathways as it degrades activated C4b and C3b when these are bound to a cofactor such as C4b-binding protein or factor H. Here, we describe a method for purification of FI from human plasma, which is based on affinity chromatography followed by anion exchange chromatography. We also describe a functional assay, in which activity of FI can be assessed.