This report describes the stege I/II development of a new direct agglutination test (DAT) for the diagnosis of canine visceral leishmaniasis (CVL) using freeze-dried antigen produced Coomassie blue-stained Leish-mania (Leishmania) infantum promastigotes. In stage I, 16 canine serum samples, collected from eight dogs carrying CVL and eight healthy dogs, were assessed with the DAT using 2-mercaptoethanol (2-ME), N-acetyl-cysteine (NAC), kaolin or NAC plus urea (NAC + U) to improve the assay conditions. Stage II assessed the diagnostic accuracy with 100 serum samples collected from dogs with symptomatic CVL and clin-ically healthy dogs, comparing the four different sample diluents. The CVL-DAT prototype kit showed equivalent performances when 2-ME, NAC or NAC + U were used: 97.1% sensitivity (CI: 83–99.8%), 97% specificity (CI: 88.5–99.5%) and a 97% diagnostic accuracy (CI: 90.8–99.2). With kaolin, a 94.1% sensitivity (CI: 79–99%), 97% specificity (CI: 88.5–99.5%) and 96% diagnostic accuracy were observed (CI: 89.5–98.7), with no statistically significant differences among the four reagents (p = 1.0). The NAC plus urea in sample diluent decreased non-specific agglutination, promoted a better defined sharp-edged blue spot and was thus chosen as a component for the new DAT prototype to diagnose canine VL, designated DAT-Canis.