Human asparagine synthetase (ASNS) catalyzes the conversion of aspartate to asparagine in an ATP-dependent reaction that utilizes glutamine as a nitrogen source while generating glutamate, AMP, and pyrophosphate as additional products. Asparagine Synthetase Deficiency (ASNSD) is an inborn error of metabolism in which children present with homozygous or compound heterozygous mutations in the ASNS gene. These mutations result in ASNS variant protein expression. It is believed that these variant ASNS proteins have reduced enzymatic activity or stability resulting in a lack of sufficient asparagine production for cell function. Reduced asparagine production by ASNS appears to severely hinder fetal brain development. Although a variety of approaches for assaying ASNS activity have been reported, we present here a straightforward method for the in vitro enzymatic analysis by detection of AMP production. Our method overcomes limitations in technical feasibility, signal detection, and reproducibility experienced by prior methods like high-performance liquid chromatography (HPLC), ninhydrin staining, and radioactive tracing. After purification of FLAG-tagged R49Q, G289A, and T337I ASNS variants from stably expressing HEK 293 T cells, this method revealed a reduction in activity of 90%, 36%, and 96% respectively. Thus, ASNS protein expression and purification, followed by enzymatic activity analysis, has provided a relatively simple protocol to evaluate structure-function relationships for ASNS variants reported for ASNSD patients.