Abstract: The present invention relates to a method for assaying transferase activity upon incorporation of an affinity ultrafiltration process as a separation means, which method comprises: (1) reacting the transferase to be assayed with a labeled substrate and an unlabeled substrate to yield a product having a label moiety from the labeled substrate and a binding site moiety either from the unlabeled substrate or existing as a specific structure of the product, (2) contacting the reaction mixture with a soluble macroligand capable of forming a specific complex with the product via the binding site moiety, (3) subjecting the complex mixture to ultrafiltration which retains the complex and passes the unreacted labeled substrate, (4) washing the retentate, and (5) determining the final retentate. The present invention also relates to a kit embodying the inventive concept of the affinity ultrafiltration assay for transferase activity.