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Cyclophosphamide exhibits the weakest therapeutic effect compared with vincristine and doxorubicin in the CHOP (C, cyclophosphamide; H, doxorubicin; O, vincristine; and P, prednisolone) chemotherapeutic protocol for the treatment of canine lymphoma. Twenty dogs with multicentric lymphoma were treated using the LHOP protocol, which used l-asparaginase in place of cyclophosphamide, and the outcomes were historically compared with those of dogs that received CHOP chemotherapy in the same institution. No significant differences were found in age (<i>p</i> = 0.107), body weight (<i>p</i> = 0.051), sex (<i>p</i> = 0.453), clinical stage V (<i>p</i> = 1), substage b (<i>p</i> = 0.573), T-cell phenotype (<i>p</i> = 0.340), overall response (<i>p</i> = 1), and hypercalcaemia status (<i>p</i> = 1) between the LHOP and CHOP groups. The adverse effects of l-asparaginase were well tolerated and self-limiting. The median PFS (progression-free survival) and median ST (survival time) in the LHOP group were 344 days (range: 28–940 days) and 344 days (range: 70–940 days), respectively. The median PFS and median ST in the CHOP group were 234 days (range: 49–1822 days) and 314 days (range: 50–1822 days), respectively. The dogs that received LHOP chemotherapy had a significantly longer PFS than the dogs that received CHOP chemotherapy (<i>p</i> = 0.001). No significant difference was observed in ST between the LHOP and CHOP groups (<i>p</i> = 0.131). Our study findings thus indicate that the LHOP protocol can be used as a first-line chemotherapeutic protocol in canine multicentric lymphoma.