Perennial ryegrass (Lolium perenne L.) is a dominant species in temperate Australian pastures. Currently, nitrogenous fertilizers are used to support herbage production for pasture and fodder. Increasing the nitrogen use efficiency (NUE) of pasture grasses could decrease the amount of fertilizer application and reduce nitrogen (N) leaching into the environment. NUE, defined as units of dry matter production per unit of supplied nitrogen, is a complex trait in which genomic selection may provide a promising strategy in breeding. Our objective was to develop a rapid, high-throughput screening method to enable genomic selection for y-60NUE in perennial ryegrass. NUE of 76 genotypes of perennial ryegrass from a breeding population were screened in a greenhouse using an automated imagebased phenomics platform under low (0.5 mM) and moderate (5 mM) N levels over 3 consecutive harvests. Significant (p < 0.05) genotype, treatment, and genotype by treatment interactions for dry matter yield and NUE were observed. NUE under low and moderate N treatments was significantly correlated. Of the seven plant architecture features directly extracted from image analysis and four secondarily derived measures, mean projected plant area (MPPA) from the two side view images had the highest correlation with dry matter yield (r = 0.94). Automated digital image-based phenotyping enables temporal plant growth responses to N to be measured efficiently and non-destructively. The method developed in this study would be suitable for screening large populations of perennial ryegrass growth in response to N for genomic selection purposes.