<p>Leptospirosis is a widespread zoonotic disease of significant veterinary and public health importance, particularly in dogs and cattle. The present study aimed to develop and evaluate recombinant Loa22 (rLoa22)-based serological assays, including an indirect enzyme-linked immunosorbent assay (iELISA) and a latex agglutination test (LAT), for the detection of anti-leptospiral antibodies in canine and bovine sera. Serum samples were initially screened using the microscopic agglutination test (MAT) to classify true positive and true negative samples. The LAT was standardized using rLoa22-coated latex beads and evaluated for diagnostic accuracy, analytical specificity, prozone effect, and shelf-life stability. Receiver operating characteristic (ROC) analysis was employed to determine optimal iELISA cut-off values, and agreement between assays was assessed using Cohen’s kappa coefficient. The iELISA demonstrated excellent diagnostic performance, with area under the curve (AUC) values of 0.988 in dogs and 1.00 in cattle, achieving 100% sensitivity and ≥ 96.81% specificity, along with excellent agreement with MAT (κ = 0.91 in dogs; κ = 0.97 in cattle). The rLoa22-based LAT exhibited 100% sensitivity and specificities of 89.36% in dogs and 91.67% in cattle, showing substantial agreement with MAT (κ = 0.79 for both species). However, the observed high sensitivities may be influenced by the limited number of MAT-positive samples. No cross-reactivity was detected with <i>Brucella</i> or enterobacterial infections, although a prozone effect was observed at high antibody concentrations. The assays were further validated using serum samples from dogs (n = 300) and cattle (n = 98) suspected of leptospirosis. Overall, the rLoa22-based LAT and iELISA may serve as useful complementary tools for the screening of leptospirosis, particularly in endemic and resource-limited settings. However, validation in a larger set of MAT-positive samples is warranted.</p>