The term impurity reflects unwanted chemicals that are present in APIs or that develop during formulation or upon aging of the API in the formulated drug product. The presence of such unwanted material, even in small amounts, could affect the efficacy and safety of pharmaceutical products. Impurities are substances that are present in small quantities in another substance and make it dirty or of an unacceptable quality. Moreover they might have adverse effects also. Identification, isolation and characterization of unknown impurity in Asenapine maleate Drug Substancesdone by HPLC, mass spectrometer and NMR. After thermal forced degradation of Asenapine maleate Drug Substances, a major unknown impurity observed in at RRT 2.0 with respect to Asenapine maleate in HPLC method. A chromatographic method with C8 column, ammonium acetate in water, acetonitrile and methanol as the mobile phase with gradient elution has been developed for LC-MS. Mass spectrometric studies of impurity conducted in electro spray ionization (ESI) mode.The ESI mass spectrum of this impurity at RRT 2.0 showed a protonated molecular ion [M+H]+ at m/z 282. The impurity isolated from preparative HPLC to get pure form and further molecular weight determined from the mass spectrometer and NMR studyperformed. From the NMR data and mass spectra, the structure of impurity elucidated and proven that the impurity formed from Asenapine maleate only. Asenapine maleate unknown thermal degradation impurity was confirmed as 5-chloro-2-methyl-2H-dibenzo[2,3:6,7]oxepino[4,5- c]pyrrole.This study and its findings will aid in ensuring the quality and safety of Asenapine maleate Drug Substances and Drug Products.