Temozolomide (TMZ) is an imidazotetrazine prodrug used to treat glioblastoma multiforme. Its physicochemical prop-erties and small size confer the ability to cross the blood-brain barrier. The antitumor activity depends on pH-dependent hydrolysis of the methyldiazonium cation, which is capable of methylating purine bases (O6-guanine; N7-guanine, and N3-adenine) and causing DNA damage and cell death. TMZ is more stable in acidic media (pH ≤ 5.0) than in basic media (pH ≥ 7.0) due to the protonated form that minimizes the catalytic process. Because of this, TMZ has high oral bioavailability, but it has a half-life of 1.8 h and low brain distribution (17.8%), requiring a repeated dos-ing regimen that limits its efficacy and increases adverse events. Drug delivery Nanosystems (DDNs) improve the phys-icochemical properties of TMZ and may provide controlled and targeted delivery. Therefore, DDNs can increase the efficacy and safety of TMZ. In this context, to ensure the efficiency of DDNs, analytical methods are used to evaluate TMZ pharmacokinetic parameters, encapsulation efficiency, and the release profile of DDNs. Among the methods, high-performance liquid chromatography is the most used due to its detection sensitivity in complex matrices such as tissues and plasma. Micellar electrokinetic chromatography features fast analysis and no sample pretreatment. Spec-trophotometric methods are still used to determine encapsulation efficiency due to their low cost, despite their low sen-sitivity. This review summarizes the physicochemical and pharmacological properties of free TMZ and TMZ-loaded DDNs. In addition, this review addresses the main analytical methods employed to characterize TMZ in different ma-trices.