Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGANPs) have been widely investigated for sustained and targeted delivery of various drugs including small molecular drugs (hydrophobic/ hydrophilic drugs) and macromolecule drugs (such as proteins, peptides, genes, vaccines, antigens, human growth factors, etc.). The in vivo pharmacokinetics and disposition profile of these encapsulated drugs and PLGANPs themselves is a key factor that determines their therapeutic index and potential for clinical use. Therefore, this review attempts to outline the in vivo behaviors of diverse drugs loaded PLGANPs administrated via different routes such as oral route, intravenous injection, nasal path, etc. Also, the associated analytical techniques used to investigate the in vivo disposition of PLGANPs loaded with drugs are focused on.