<p>Microtubule affinity-regulating kinase (MARK), particularly MARK4, are involved in the pathological phosphorylation of tau, contributing to neurodegenerative diseases and conditions such as cancer, inflammation, and atherosclerosis. The β-carboline family, specifically Harmane, exhibits broad biological activity, including neuroprotective effects. We investigated the inhibitory potential of Harmane against MARK4 using both computational and experimental approaches. The interaction between Harmane and MARK4 was studied using a combination of computational and experimental approaches. Molecular docking was carried out to understand the binding of Harmane to the binding pocket of MARK4, focusing on key interactions. Molecular dynamics simulations further assessed the stability of the MARK4-Harmane complex. Enzyme inhibition assays were conducted to assess Harmane’s inhibitory potential on kinase, and a fluorescence quenching assay was complemented to validate the binding affinity of Harmane with MARK4. Molecular docking revealed that Harmane binds to the active site of MARK4, a finding supported by molecular dynamics simulations, demonstrating increased stability of the MARK4-Harmane complex. Structural analysis further highlighted the specificity of this interaction. Enzyme inhibition assays estimated Harmane’s IC₅₀ (half-maximal inhibitory concentration) value as 2.72 µM against MARK4, while fluorescence spectroscopy measured a binding constant (<i>K</i>a) of 0.1 × 10⁵ M^− 1. These results strengthen the idea that Harmane can be a potent MARK4 inhibitor, offering therapeutic promise for neurodegenerative diseases and cancer.</p>