Currently available approaches to screen resistance of various Prunus (stone fruit) rootstocks to Armillaria spp. may only provide partial evidence for resistance and are labor and time-intensive. Our objective was to overcome the limitations of the available methods and develop a holistic in vitro assay to screen tolerance/susceptibility of Prunus spp. to Armillaria spp. Sterile root segments of three Prunus spp. were placed next to or on top of 14-day-old cultures of A. solidipes. At 21-days after inoculation, the success of fungal penetration, the length of the fungal colonization, and host responses were evaluated. Also, an anti-Armillaria and antifungal compound detection assays were conducted by utilizing the root periderm, the first tissue that needs to be penetrated by the fungus. These methods revealed the variation in the relative tolerance/susceptibility of three Prunus spp. to A. solidipes. The overall success of fungal colonization in the wounded and intact root, host defense, and antifungal activity significantly differed among various Prunus spp. This holistic screening technique allowed associated active and passive host defenses to be assessed in three weeks. Results suggest that host tolerance/susceptibility to Armillaria spp. relies on the active and passive defenses of mature root periderm and this in vitro method can be used as a preliminary step in screening tree species to Armillaria spp.