Myrosinase (thioglucoside glucohydrolase; EC 3.2.3.1) is an enzyme that hydrolyzes glucosinolates to D ‐glucose and allelochemicals that have biological potential to suppress weed seed germination in soil. This enzyme, found in some microorganisms and released to soils via root exudation and decomposition, can be assayed by adding sinigrin (2‐propenyl‐glucosinolate) to soil as a substrate. We describe a simple and rapid method to assay myrosinase activity in soils. In this method, 1 g of soil is treated with toluene (0.2 mL) and incubated at 37°C with 2.8 mL of a buffered solution (pH 7) of sinigrin (20 m M final concentration) for 4 h. Glucose released upon sinigrin hydrolysis is extracted and its concentration is measured spectrophotometrically. Tests showed that recovery of glucose was quantitative if toluene was included in the assay mixture. Myrosinase activity in five soils studied ranged from 71 to 338 μg glucose g −1 soil 4 h −1 The rate of sinigrin hydrolysis increased with temperature from 10 to 40°C. The activation energy of myrosinase in four soils ranged from 40.3 to 52.8 kJ mol −1 The V max values for sinigrin hydrolysis calculated by the three linear transformations of the Michaelis–Menten equation ranged from 76 to 518 (avg. 275) μg glucose g −1 soil 4 h −1 and the apparent K m values for myrosinase ranged from 5.3 to 12.9 (avg. 8.1) m M The method developed in this study is accurate, fast, and simple.