In light of its superior metabolic stability, the thiophosphate motif is widely regarded as a valuable substitute for its native phosphate counterpart1–3. This property has led to the incorporation of thiophosphates and phosphorothioates in various therapeutic modalities, such as antisense oligonucleotides4,5 and cyclic dinucleotide analogs6. Biochemically, thiophosphates can be installed on proteins by combining protein kinases and adenosine-5′-O-(3-thio-triphosphate) (ATPγS)7, but the method has never been explored on additional substrate classes and is impractical to scale up because it requires superstoichiometric amounts of the expensive ATPγS. Here, we report the invention of an ATPγS recycling strategy, which circumvents these limitations through the use of a specially designed creatine derivative to turn over the kinase reaction byproduct. The developed protocol significantly improves the practicality of enzymatic thiophosphorylation and is compatible with many kinases. The versatility of the strategy is demonstrated in the design of multi-enzyme cascades to synthesize a diverse range of thiophosphate-containing small and macromolecules, including nucleoside 5′-monothiophosphates, 3′,5′-cyclic monophosphorothioates and thiophosphorylated oligopeptides. Our findings open the door for new biocatalytic approaches to thiophosphate-containing modalities in drug development endeavors.