Background Rabacfosadine (RAB), a novel antineoplastic agent conditionally licensed for the treatment of lymphoma in dogs, is efficacious in both naive and previously treated dogs. Its use in combination with L-asparaginase (L-ASP) has not been studied. Hypothesis/objectives To evaluate the safety and efficacy of L-ASP given concurrently with RAB in dogs with relapsed multicentric lymphoma. Animals Fifty-two dogs with relapse of lymphoma after treatment with at least 1 doxorubicin-based chemotherapy protocol. Methods Open-label, multicenter, prospective single-arm clinical trial. Dogs were treated with RAB at 1.0 mg/kg IV every 21 days for up to a total of 5 doses. L-asparaginase was administered at 400 IU/kg SQ concurrently with the first 2 treatments of RAB. Results The overall response rate (ORR) for all dogs was 67%, with 19 dogs (41%) achieving a complete response (CR). The median progression-free survival time (MPFS) was 63 days (range 5-428 days). Dogs experiencing a CR as their best response had an MPFS of 144 days (range 44-428 days). Adverse events were similar to previous studies evaluating single agent RAB. Failure to achieve a CR and having previously received L-ASP were negative prognostic factors on multivariate analysis. Conclusions and clinical importance Concurrent RAB/L-ASP appears to be both efficacious and safe for treating relapsed multicentric lymphoma in dogs. Adverse events were most often mild and no unexpected toxicoses were observed.