Summary For efficient use of available synthesised nematicides, bioassay-guided analysis is required to understand the behaviours of nematicides. In this study, the key nematicides on the market, including avermectin B1a, avermectin B2a, emamectin benzoate, fluensulfone, fluopyram and fosthiazate, were evaluated by both in vitro and in planta approaches with optimised solvent and nematode inoculum. We found that acetone was the only one of the six solvents tested safe for both root-knot nematode (RKN) second-stage juveniles (J2) and cucumber at the tested concentration. The optimal numbers of RKN J2 for inoculation on cucumber seedlings in the glass-tube assay, cup assay and microplot experiment were 200, 700 and 1250, respectively. No obvious phytotoxicity symptoms were observed in the in planta assays for all nematicides selected at the tested concentration in the soil drench application. When the substrate changed from pure sand to matrix-sand, the EC50 and EC90 of all six nematicides increased. Among them, fluopyram rose the most. In the microplot experiment, avermectin B2a showed the lowest EC50 but the EC90 of avermectin B2a, fluensulfone, fluopyram and fosthiazate were similar. The results are useful as a guide to the application of commonly used nematicides in the field.