Background: Breast cancer is the most common cancer diagnosis and the second leading cause of cancer-related death in women. Breast cancer is a major health burden worldwide. Advances in breast cancer detection and treatment have contributed to improving the rate of survival, although mortality rates remain significantly high. Despite all these advances, more efficient diagnostic methods and effective treatments are necessary. Colchicine is a natural alkaloid with strong antimitotic activity, but its potential effects on extracellular matrix components in cancer remain poorly understood. Objective: This study aimed to investigate the influence of colchicine on glycosaminoglycan (GAG) concentrations and cell viability in MCF-7 breast cancer cells cultured in a three-dimensional (3D) hollow fiber bioreactor system. Methods: Magnetic resonance imaging (MRI) was applied as a non-invasive technique to quantify GAG levels through fixed charge density (FCD) and T1 relaxation mapping. MCF-7 HER-2-overexpressing and HER-2-negative cells were treated with 1000 nM colchicine for 72 h, and cell viability was assessed in parallel with GAG measurements. Results: Colchicine significantly reduced cell viability and altered GAG concentrations. HER-2-overexpressing MCF-7 cells exhibited higher baseline GAG levels than HER-2-negative controls, and colchicine decreased the GAG content in both lines. Conclusions: Colchicine reduces viability and modifies GAG concentrations in 3D cultures of MCF-7 cells. The use of MRI provides a reproducible, non-destructive tool for monitoring extracellular matrix changes, offering a novel methodological approach for studying drug effects in physiologically relevant cancer models.