Ticks may contain a diverse array of both pathogenic and invertebrate-specific viruses. Culture methods can be performed in vitro or in vivo to isolate and culture these viruses. Here, we demonstrate the culture of Deer tick virus (DTV) and Powassan virus (POWV) from ticks collected in Upstate New York. Research into medically significant tick-borne pathogens can be facilitated through in vitro models using cultured mammalian cells. This is especially true of viruses, which require amenable host cells to replicate. This chapter describes the isolation of viruses from field-collected ticks utilizing basic mammalian cell culture techniques.