Extracellular enzymes break down soil organic matter into smaller compounds and their measurement has proved to be a powerful tool to evaluate the functionality of soils. Urease is the enzyme that degrades urea and is widely considered to be a good proxy of nitrogen (N) mineralisation. But the methods available to measure this enzyme are time consuming; as such, urease is not commonly included in standard enzyme profiling of soils. We developed a fast, high throughput and reproducible colorimetric microplate technique to evaluate urease activity in soil. The method involves the incubation of soil slurries in 96-deepwell blocks with urea solutions and the measurement, by colorimetric reaction, of ammonium produced. We compared the new method with existing methods, yielding comparable results, and evaluated optimal conditions for urease analysis (soil slurry concentration, substrate concentration, incubation times and extractant salt concentration) in different grassland soils. The method proved to be a faster, higher throughput, and more precise alternative to existing methods for evaluating this important N-related enzyme.