Stabilization of G-quadruplex structures formed from telomeric DNA, by means of G-quadruplex selective ligands, is a means of inhibiting the telomerase enzyme. This makes G-quadruplex an emerging target for cancer therapy. The objective of the current 3D QSAR study is to uncover structural requirements for acridine derivatives, which would eventually assist and complement the rational drug-design attempts. Various protonation strategies were investigated to check in situ protonation sites present on ligands when they bind to G-quadruplex, and predictive 3D-QSAR CoMFA and CoMSIA models have been developed. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) studies were carried out on substituted acridines as telomerase inhibitors. Molecular models with good predictive power were derived using steric, electrostatic, hydrophobic, and H-bond donor fields of the compounds. The CoMSIA coefficient contour plots identified several key features explaining the wide range in activities. The present study not only offers a highly significant predictive CoMSIA model for trisubstituted acridine derivatives as telomerase inhibitors but also throws more light on the molecular structure of these compounds at physiological pH.