B-Cell Acute Lymphoblastic Leukemia (B-ALL) is a subtype of leukemia characterized by the uncontrolled proliferation of B-lymphocytes. The diagnosis of B-ALL relies on blood counts, immunophenotyping, cytogenetic analysis, and molecular tests. Treatment strategies for B-ALL include chemotherapy, radiotherapy, allogeneic bone marrow transplantation, and stem cell transplantation. In the diagnosis and treatment of B-ALL, antibodies or antibody fragments specific to CD19, a protein known as a B-lymphocyte antigen, are commonly used. However, producing antibodies specific to targeted antigens is often cost-prohibitive. Nanobodies, which constitute the heavy chain variable region of heavy-chain only camelid antibodies have been elucidated to be more advantageous. Three nanobodies (Nbs), which were chosen from a natural Nb-expressing phage display library in the study of (H. Wang et al. 2021a), with affinity to CD19 were tested. The nanobodies, which are derived from the heavy-chain antibodies of camelids and constitute the single-domain heavy variable region of the antibodies, were maturated in-silico. Two parent nanobodies, out of three, were chosen alongside four variants based on their in-silico performance. The purpose of this study is to verify the applicability of our in-house affinity maturation protocol (structure prediction, filtering, and scoring), by in-vitro production and functional characterization of affinity maturated variants against their parents nanobodies. Moreover, it is reflected on multiple paradigms concerning in-silico affinity maturation of nanobodies, and how models fare under the reality of experimentation.