Puccinia striiformis, the causal agent of stripe rust disease, poses a significant threat to cereal crops worldwide. Since 2000, the emergence of heat-adapted races/lineages of the wheat-infecting form P. striiformis f. sp. tritici (Pst) has enabled the disease to spread beyond its traditional high-elevation, cool, and moist regions. It can now occur wherever wheat is cultivated, making it a pandemic concern. This chapter provides an overview of the methodologies and practices established in our lab for working with this pathogen. These methodologies include isolate collection, recovery, purification, increase, and storage, as well as the evaluation of virulence in a greenhouse setting, and the fundamentals of running a large-scale yearly disease nursery under field conditions. This comprehensive guide, based on our lab’s experience with stripe rust, aims to provide researchers with the knowledge and tools necessary for effective and informed studies on P. striiformis.