As tick-borne viruses such as Powassan virus (POWV) and Deer tick virus (DTV) increase in prevalence, having methodology to isolate circulating strains from field-collected ticks grants an opportunity to better understand viral evolution, to more accurately assess human health risks, and to make research into these viruses more translational. POWV and DTV are genetically similar but distinct emerging tick-borne viruses in North America that can cause severe encephalitic disease with high morbidity and mortality. Research into these viruses typically use prototypical lab strains, which may not reflect characteristics of circulating populations of these viruses. Here we will outline a protocol for reliably isolating DTV and POWV from field-collected ticks using an in vivo system.