The number of novel mycoviruses is increasing at a high pace due to advancements in sequencing technologies. As a result, an uncountable number of mycoviral sequences are available in public sequence repositories. However, only genomic information is not sufficient to understand the impact of mycoviruses on their host biology. Biological characterization is required to determine the nature of mycoviruses (cryptic, hypervirulent, or hypovirulent) and to search for mycoviruses with biocontrol and therapeutic potential. Currently, no particular selective method is used as the gold standard against these mycoviral infections. Given the importance of curing, we present an overview of procedures used in preparation of isogenic lines, along with their benefits and drawbacks. We concluded that a combination of single-spore isolation and hyphal tipping is the best fit for preparation of isogenic lines. Furthermore, recent bioinformatic approaches should be introduced in the field of mycovirology to predict virus-specific antivirals to get robust results.