Lithiation-substitution protocol is a significant tool for the formation of a new C-C bond in heteroatom-containing compounds. This strategy has been extensively explored to synthesize many heterocyclic drugs and drug intermediates. These reactions are always considered very complex and many research groups have worked to explore the regioselectivity and stereoselectivity of these reactions. The feasibility of these reactions is affected by many factors like the nature of the lithiation reagent, solvent and temperature. In this chapter, we will discuss in detail the different lithiating reagents, the effect of solvent, temperature, regioselectivity and stereoselectivity. This chapter will also deal with the different mechanistic aspects involved in the regioselective and stereoselective outcomes of the lithiation-substitution reaction.