More than 80% livestock are facing severe problems of ticks and tick-borne diseases (TTBDs), which create remarkable capital loss to animal owners in terms of direct blood feeding and indirectly by transmitting pathogens. Due to the overburden of synthetic chemicals in the management of TTBDs, tick populations have developed acaricide resistance against commonly used chemicals. Laboratory-based monitoring tools have been developed and adopted in different countries for the diagnosis of acaricidal resistance and its monitoring. The Food and Agriculture Organization of the United Nations recommends in vitro tests such as larval packet test (LPT), larval immersion test (LIT), and adult immersion test (AIT). Similarly, resistance diagnosis is developed using a multiplex polymerase chain reaction (PCR), which facilitates the detection of variation in nucleotide substitutions responsible for conferring resistance to synthetic pyrethroids in tick population. PCR diagnostic assays are used to identify the resistance-associated mutation in different regions of the targeted gene. To understand the involvement of metabolic enzymes in the development of resistance, biochemical tests using tick larvae are also developed and are commonly used methods. Thus, in this chapter, various in vitro tools for monitoring acaricide resistance in tick populations are discussed.