The present invention relates to a method of sterilizing a chromatography column which is packed with a separation matrix, which method comprises passing a superheated aqueous liquid through the packing of the column to obtain at least one substantially sterile packed chromatography column. The method may also comprise a preceding heating. In the most advantageous embodiment, the superheated aqueous liquid is passed in a substantially uniform flow through the packing of the column, resulting in a sterility of the packed column.