The present invention relates to a two-step method for isolating proteins from the cystine-knot superfamily based on dye ligand affinity chromatography and reversed-phase chromatography. Advantageously, the method can be performed in a relatively short period of time, involves inexpensive reagents, and requires little sample preparation before and during the purification process. Protein fusions between cystine-knot proteins and proteins of interest are further provided for the isolation of said protein of interest or complexes containing said protein of interest using the two-step method disclosed.