Technical Abstract: The relative instability associated with plant Asparagine synthetase (AS) has made it difficult to extract and assay this enzyme in an active form. We present an improved extraction prototol yielding highly active AS together with an easy and reproducible enzymatic assay using radiolabelled [14C]aspartate to monitor in vitro asparagine formation in crude plant extracts. We optimized conditions for the preparation of crude extracts containing plant AS, and developed a rapid and quantitative HPLC method to measure de novo asparagine synthesis. Optimal [14C]asparagine production was attained after two hours incubation at 25 oC. Under the conditions of this assay, maximum activity was achieved with a concentration of 1 mM glutamine in the assay buffer. This new extraction protocol increased the storage half-life of this enzyme to approximately 9 to 10 d when stored at -25 oC. Using this extraction and assay protocol, AS retains 80% of its activity within the first three days. Activity was measurable for up to twelve days.