Puccinia coronata (crown rust) is a damaging pathogen of ryegrasses used for both forage and turf applications. Alternative sources of crown rust pathogen resistance with enhanced durability are required for perennial ryegrass breeding programs. This approach may require pyramiding of major and minor resistance gene combinations into varieties, opposing the capacity of evolving pathogen populations to overcome complex resistance mechanisms. Crown rust pathogen populations have been homogenised into distinct lineages on detached leaves through the use of molecular genotyping technology. Subsequent propagation and inoculation of such spore lineages on defined host populations and individuals requires high inoculation efficiency, given the delivery of initially small spore quantities. In this study, an inoculation protocol was developed for effective uniform infection on susceptible host plant genotypes at both seedling and adult developmental stages. A randomised complete block design was used to test the protocol on glasshouse-grown plants from 10 elite perennial ryegrass cultivars. Significant quantitative differences in pustule number and latency period were detected between cultivars, suggesting that the protocol was highly effective at providing a uniform distribution of pustules in low and high urediniospore suspension volumes. The inoculation and quantification methods provide a framework for standardised assessment of crown rust infection under controlled environment conditions, and may in principle be applied to any Puccinia—host interaction.