In green house conditions, an improved protocol for testing barley plants against root lesion nematode (RLN) had been standerdized.  The plants were grown in 150 cm³ instead of 20 cm³ tubes and we increased the inoculum size from 400 to 1000 nematodes/plant in combination with a nutrient solution better adapted to the barley crop. Six barley accessions were tested with Pratylenchus neglectus and Pratylenchus penetrans. Tests were evaluated 7, 8, 9, and 10 weeks after inoculation. It was found; a shorter test period of 7 weeks is suitable for distinguishing resistant and susceptible genotypes. It was further demonstrated that 7 weeks after inoculations is sufficient to determine nematode numbers instead of 10 or 12 weeks after inoculation. Moreover, measuring the root fresh weight was necessary to calculate the relative number of nematodes/g root mass. In conclusion, results led to a more efficient, quicker and more accurate measurement of barley infections by Pratylenchus spp. which is important for selection during early generations of cultivar development.