A process has been developed for separation of plutonium from uranium in the partitioning cycle of reprocessing of fast reactor fuel using a new salt free reagent acetohydroxamic acid (AHA). Acetohydroxamic acid is used as a partitioning agent based on its novel behaviour of reductant/complexant. AHA selectively forms a complex with plutonium than uranium which facilitates stripping of plutonium to the aqueous phase leaving uranium in the organic phase. The effect of plutonium and uranium concentration on feed organics and a number of contacts are investigated in order to maximize the separation factor. It is found from the data that almost 99% of plutonium is stripped into the aqueous phase leaving >93% of uranium in the organic phase itself by just giving three contacts.