Earthworms are target organisms both for scientists studying the biological component of soils and for farmers concerned with monitoring the quality of their soils. Different expellants are used to extract earthworms from the soil but differences in chemical properties and efficiency between commercial mustard and allyl isothiocyanate (AITC) solutions remain unknown. The objectives of this study were to compare (i) the concentration of irritating product (allyl isothiocyanate AITC) in two expellant solutions (diluted mustard or AITC solution) and (ii) their efficiency in extracting earthworms from the soil. AITC concentration was analyzed according to a new method, based on AITC solvent extraction and HPLC quantification, in one commercial mustard brand to assess its variability within and between batches of jars. According to mustard spiking with AITC standard solution, extraction recovery was estimated as 98 ± 2%. Earthworm field data were collected in spring 2012 in 22 cultivated fields located in east Île-de-France, comparing pure AITC to commercial mustard solutions. Species diversity, abundance and biomass of earthworms per plot were measured. We showed that AITC concentration in commercial mustard varied according to the use by date but not according to the batch. We thus recommend using the freshest mustard available from the same batch. Moreover, AITC solution was found to be about four times more concentrated in AITC than the commercial mustard solution. Despite this result, no significant differences were found in the efficiency of commercial mustard or AITC solutions to bring earthworms to the soil surface in terms of abundance, biomass or diversity. We thus discuss the advantage and drawbacks of using both expellants in the field.