We recently used RNA interference to show that a negative correlation of l-asparaginase (l-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and l-ASP activity using an MTS assay on 19 human ovarian cancer cell lines. Contrary to our previous findings, l-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson's correlation coefficients were r = -0.21 for microarray data and r = -0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (P = 0.047, one-tailed). ASNS protein expression measured by liquid-phase immunoassay exhibited a much stronger correlation (r = -0.65; P = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of l-ASP activity in ovarian cancer cell lines. These findings provide rationale for evaluation of ASNS protein expression as a predictive biomarker of clinical l-ASP activity in ovarian cancer. [Mol Cancer Ther 2008;7(10):3123–8]