Background: l-Asparaginase hydrolyzes l-asparagine and not its enantiomer d-asparagine. Unlike l-asparagine, d-asparagine is nonessential for the survival of acute lymphoblastic leukemia (ALL) cells. Studies showed that serum asparagine is depleted below 0.5 μM in ≥96% of the patients during pegylated Escherichia coli l-asparaginase (PEGasparaginase) treatment; however, cerebrospinal fluid (CSF) asparagine levels are depleted in only 20%–30% of the patients. Thus far, studies only reported the total CSF asparagine (sum of d- and l-asparagine) concentrations. Data on the pharmacological goal, which is l-asparagine depletion, are lacking. Method: Therefore, we studied this in 30 patients (95 samples) with newly diagnosed ALL. They received two doses of PEGasparaginase on day 4 and 18 in induction. Results: Median age at diagnosis was 5.7 years (range 1.5–17.1 years). d-Asparagine and l-asparagine concentrations (median (range)) before PEGasparaginase treatment were 0.038 (0.0–0.103) μM and 6.1 (1.82–11.5) μM, respectively. CSF l-asparagine concentrations were reduced by 85% (76%–100%) and approximately one-third of the patients (32%) had CSF l-asparagine depletion below 0.5 μM 11 days after the second PEGasparaginase dose administration. CSF d-asparagine and l-glutamine levels remained stable before and after administration of PEGasparaginase. The percentage of d-asparagine as a fraction of total asparagine (sum of d- and l-asparagine) was 0.62% before and 4.5% after PEGasparaginase treatment. No correlation was found between higher serum PEGasparaginase activity and CSF l-asparagine concentration. Conclusion: l-Asparagine is not a better parameter than total asparagine in CSF due to the negligible amount of d-asparagine in the CSF before and after PEGasparaginase treatment.