Formation of G-quadruplex in the telomeric region of human chromosome followed by its stabilization with small ligands, is a prominent strategy for telomerase inhibition. Interaction of human G-quadruplex with antitumor anthraquinone derivative mitoxantrone (MTX) has been studied by multi-spectroscopic techniques. Presence of clear isobestic point in absorption spectra at 677 nm indicates existence of a single mode of interaction with a binding constant = 2.23 x 105 M-1. Titrations monitored by fluorescence give binding stoichiometry of MTX-d-(TTAGGGT)4 as 2:1. Thermal stabilization of G-quadruplex with an increase in Tm of 6◦C is observed on binding by monitoring circular dichroism spectra. The exact mode of interaction has been revealed by proton and phosphorus-31 Nuclear Magnetic Resonance spectroscopy. The imino protons disappear at higher temperatures in the complex as compared to uncomplexed DNA. The observed intermolecular Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and sugar protons of G-quadruplex indicate that the ligand binds at the DNA groove. Dose dependent telomerase inhibition has been observed by Telomere Repeat Amplification Protocol (TRAP) assay at EC50 = 2 μm. The present investigations lead to the conclusion that mitoxantrone can be a promising G4 ligand. The research work is funded by DST, Govt. of India.