Toxoplasma gondii is an intracellular heteroxenous protozoan parasite that causes toxoplasmosis in warm-blooded vertebrates, globally. This parasite is widespread in humans and other animal species and has already been reported in many countries and diverse climates. The main aims of this study were to assess the seropositivity to toxoplasmosis of exotic breed of cattle (n = 400) from different farms by using Latex Agglutination Test (LAT), validation of newly developed ELISA kit (Cattle Toxo IgG ELISA Kit) with the help of commercially available ELISA kit available as “ID Screen Toxoplasmosis Indirect Multi-species ELISA Kit”(taken as Gold Standard in the current study) and comparative efficacy of LAT and newly developed ELISA kit for assessing seropositivity of cattle to toxoplasmosis. Out of a total of 400 cattle sera that were evaluated by LAT, 143 (35.75%) were found positive. Based on this data, 90 samples (n = 60 seronegative by LAT; n = 30 seropositive by LAT) were selected for screening through Gold Standard ELISA kit. The same 90 samples were screened through our newly developed ELISA kit for validation purpose. Out of 40 samples found seropositive in our newly developed ELISA kit (100% sensitivity), 38 were also seropositive in Gold Standard ELISA kit. Whereas all the 50 samples found seronegative in our newly developed ELISA kit (96.15% specificity), were also seronegative in Gold Standard ELISA kit. Hence the sensitivity and specificity of our newly developed ELISA kit came out to be 100% and 96.15%, respectively. The sensitivity and specificity of LAT in the current study was found as 26.31% and 61.53%, respectively. Screening of 400 cattle sera through our newly developed ELISA kit revealed 29.75% (119/400) seropositivity, 6% less than that found through LAT. The results of this study show that our newly developed ELISA kit is highly accurate and reliable serodiagnostic tool to diagnose bovine toxoplasmosis. It is important to highlight that overall highest seropositivity was seen in cattle of age-group “5 years or above”.