A c c e p t e d M a n u s c r i p t On-chip chemotaxis assay method of plant-parasitic nematodes was developed. A protocol to allow the nematodes to move around in the microchannels was achieved. Potassium nitrate stimulated the behavior of plant-parasitic nematode M. incognita. Potassium nitrate caused attraction or repulsion depending on its concentration. Abstract 25 Plant-parasitic nematodes substantially damage a wide variety of agricultural crops 26 worldwide. The chemotaxis of nematodes is a key factor in their parasitic relationship with 27 plants. Therefore, studies on nematode chemotaxis are essential for devising efficient and 28 environmentally friendly management methods. In this paper, we report a new, efficient, and 29 quantitative method to analyze chemotaxis of the plant-parasitic nematode, Meloidogyne 30 incognita, using a gel-filled microchannel device. We quantitatively defined time-dependent 31 concentration gradients of chemicals in the gel-filled microchannel by measuring changes in 32 the fluorescence intensity of fluorescein. We also developed a protocol to allow the 33 nematodes to move around in the microchannels by loading an agarose gel with optimum 34 concentration. Using this novel assay method, we have shown that potassium nitrate (KNO 3 ) 35 stimulates the behavior of M. incognita and can have both repellent and attractant effects, 36 depending on the concentration gradient. This newly developed quantitative chemotaxis assay 37 method can be used to screen and identify new candidate molecules that repel or attract 38 nematodes, and also to analyze the repellent/attractant properties of those molecules towards 39 nematodes. 40 41 42