Background: Pronase treatment reduces nonspecific binding in B cell flow cytometric crossmatch (B-FCXM). Higher concentration of pronase might reduce false positivity from rituximab, but also can decrease the sensitivity. We evaluated the effect of variable pronase concentration on B-FCXM with sera from patients with various conditions. Methods: We analyzed 63 sera, including 30 from patients with rituximab treatment before 7–63 days ago (17 with donor-specific antibody [DSA] [MFI 1,260–10,325 for A, B, DR, and 5,226–19,033 for DQ] and 13 with non-DSA). Controls comprised 29 sera from patients with DSA but nonrituximab-treated. Additionally, we spiked 4 sera from DSA-negative and nonrituximab-treated patients with rituximab (Mabthera, Roche; 100 ug/mL). We isolated peripheral blood mononuclear cells from 38 kidney trans-plantation donors (Seoul National University Hospital, June 2022 to January 2023) and treated them with six different pronase concentrations (0, 0.5, 1.0, 2.0, 3.0, and 4.0 mg/mL). Donor cells underwent crossmatch with three patient groups: rituximab and DSA (RD, n=21), rituximab without DSA (RN, N=13), and DSA with no rituximab (ND, n=33). NDs DSA matched those of RDs specificity and MFI. The days between rituximab and blood sampling were matched between RD and RN. Results: We tested 40 DSA (six human leukocyte antigen [HLA]-A, 15 HLA-B, 12 HLA-DR, 7 HLA-DQ). The false positive rates in RN with 2.0 mg/mL (12/38, 31.6%), 3.0 mg/mL (2/38, 5.3%), and 4.0 mg/mL (0/38, 0.0%) pronase were significantly lower than 1.0 mg/mL (30/38, 78.9%) in B-FCXM (P<0.001). Sensitivity in ND with 3.0 mg/mL (23/38, 60.5%) and 4.0 mg/mL (22/38, 57.9%) pronase did not differ significantly from 1.0 mg/mL (25/38, 65.8%; P>0.05). Conclusions: Higher pronase concentration can effectively reduce false positive results in B-FCXM for patients with rituximab treatment and DSA, without a significant decrease in DSA detection sensitivity.