Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations.