A recent study suggested that mesenchymal cells in bone marrow (BM) may counteract l-asparaginase (L-Asp)-containing acute lymphoblastic leukemia (ALL) therapy by secreting asparagine. Herein, we compared asparagine and aspartic acid concentrations in the BM and peripheral blood (PB), in order to determine whether this in vitro observation could be translated into in vivo differences of amino acid levels between both compartments. Asparagine and aspartic acid concentrations in BM (days 15 and 33) and PB (days 12, 15 and 33) were measured during L-Asp-containing Berlin-Frankfurt-Münster (BFM)-based 5-week multi-agent remission induction therapy in 11 children diagnosed with ALL at the St. Anna Children's Hospital in Vienna, Austria. The level of asparagine depletion did not differ significantly between both compartments at any time point measured, but aspartic acid concentrations were significantly higher in BM than PB at days 15 and 33 (p < 0.05). In the context of the reported mesenchymal asparagine production in BM, an increased asparagine production may indeed take place in BM. However, it may be overcome by continuous action of L-Asp, which is mirrored by increased aspartic acid levels but unchanged low asparagine levels in BM, suggesting a higher BM turnover of asparagine generated by L-Asp during induction therapy.