Plant-pathogen interactions are controlled by a multilayered immune system, which is activated by pathogen recognition in the host. Pathogens secrete effector molecules to interfere with the immune recognition or signaling network and reprogram cell structure or metabolism. Understanding the effector repertoires of diverse pathogens will contribute to unraveling the molecular mechanism of virulence and developing sustainable disease-control strategies for crops and natural ecosystems. Effector functionality has been investigated extensively in only a small number of pathogen species. However, many more pathogen genomes are becoming available, and much can be learned from a broader view of effector biology in diverse pathosystems. The purpose of this review is to summarize methodology for computational prediction of protein effectors, functional characterization of effector proteins and their targets, and the use of effectors as probes to screen for new sources of host resistance. Although these techniques were generally developed in model pathosystems, many of the approaches are directly applicable for exploration and exploitation of effector biology in pathosystems that are less well studied. We hope to facilitate such exploration, which will broaden understanding of the mechanisms that underpin the biological diversity of plant-pathogen interactions, and maximize the impact of new approaches that leverage effector biology for disease control.