The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-$\beta$-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK$\alpha$, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKK$\alpha$ by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKK$\alpha$ and IKKtide, a 20 amino acid peptide substrate derived from IkB$\alpha$ kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKK$\alpha$ and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK$\alpha$ kinase activity with an IC₅₀ of 0.5 mM, to decrease the K_m value from 0.337 mM to 0.402 mM and the V_max from 0.0257 mM·min${}^{-1}$ to 0.0076 mM·min${}^{-1}$. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK$\alpha$ as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K_m and K_i whereas there is a statistically significant difference between the two V_max values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.