The stem nematode, Ditylenchus dipsaci, causes severe damage in sugar beet. To date, nematode inoculation through the leaf axil has been used as the standard method to investigate D. dipsaci interaction with sugar beet under in vivo conditions. To get as close as possible to field conditions, we established a new screening mechanism to perform soil inoculation. The most suitable inoculation time point, inoculum level and positioning on sugar beet, as well as rearing process on carrots, were determined. At a 15:8°C day:night temperature regime, penetration rates of D. dipsaci were at maximum following soil inoculation at plant emergence. Up to 115 nematodes penetrated sugar beet seedlings 22 days post-planting with an inoculum level of 1000 nematodes into the soil at plant emergence. Ditylenchus dipsaci penetration rate was higher in plants with soil inoculation than with inoculation on to the leaf axil. High soil moisture increased nematode migration into seedlings when D. dipsaci inoculation was carried out in four holes 1 cm from the plant base. Rearing the nematodes for 35 days at 20°C on carrot discs resulted in an infective inoculum containing up to 50% eggs. We recommend a soil inoculation of 1000 freshly extracted nematodes per pot at plant emergence. The nematode suspension has to be previously reared for 35 days on carrot discs to obtain active D. dipsaci inoculum. This system will allow for the selection of suitable sugar beet genotypes that suppress nematode penetration, in support of breeding for resistance against D. dipsaci.