Stem rust (caused by Puccinia graminis) and stripe rust (caused by P. striiformis) are destructive diseases of cereal crops. Newly emerged virulent races continue to pose threat to cereal productions worldwide. These heteroecious rust fungi use Berberis spp. as the alternate host to complete their life cycles. Thus, detecting new virulence combinations from aecial infections on the alternate host is an important part in rust surveillance. We have used three methods of inoculation to derive isolates of P. graminis f. sp. tritici from aecial infections on barberry plants. Method 1 is to suspend barberry leaf tissues bearing aecial pustules over seedling plants of cereal species. Under high humidity conditions, this method allows aeciospores to precipitate onto leaf surface to produce infection on the telial hosts. Method 2 is to collect and suspend released aeciospores into water and spray the inoculum suspension onto seedling of cereal hosts. Method 3 is to collect and suspend dried aeciospores into a mineral oil and spore suspension is applied onto cereal seedlings. Using these inoculation methods, we have successfully obtained many uredinial isolates from inoculations using aeciospores. Successful inoculation using aeciospores has helped us to determine the functionality of the Berberis spp. in stem rust pathogen variations and disease epidemiology in some areas where the roles of the alternate host are poorly understood.