The B-cell coreceptor, CD19 is a transmembrane protein expressed throughout B-cell ontogeny from pro-B cell to plasmablast. It plays an important role in B-cell development and function and is an attractive target for antibody-directed immunotherapies against B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (B-NHL) in humans. With the rapid development of next-generation immunotherapies aimed at improving therapeutic efficacy, there is a pressing need for a clinically relevant, immune-competent, spontaneous animal model to derisk these new approaches and inform human immunotherapy clinical trials. Pet dogs develop spontaneous B-cell malignancies, including B-NHL and leukemias that share comparable oncogenic pathways and similar immunosuppressive features to human B-cell malignancies. Despite treatment with multiagent chemotherapy, durable remissions in canine B-NHL are rare and most dogs succumb to their disease within 1 year of diagnosis. Here we report the development and validation of an anti-canine CD19-targeting monoclonal antibody and its single-chain derivatives, which enable next-generation CD19-targeted immunotherapies to be developed and evaluated in client-owned dogs with spontaneous B-NHL. These future in vivo studies aim to provide important information regarding the safety and therapeutic efficacy of CD19-targeted mono- and combination therapies and identify correlative biomarkers of response that will help to inform human clinical trial design. In addition, development of canine CD19-targeted immunotherapies aims to provide better therapeutic options for pet dogs diagnosed with B-cell malignancies.