In the present work, 15 new N'-(arylidene)-4-(1-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2-yl)benzohydrazide (4a-4o) were designed and synthesized. The structures of the synthesized compounds were elucidated using FT-IR, 1H-NMR, 13C-NMR, and HRMS spectral data. The inhibitory activity of the compounds 4a-4o against hMAO-A and hMAO-B enzymes was evaluated by using in vitro Amlex Red® reagent based fluorometric method. Due to lots of high-cost kits including this assay, we determined the ingredients of the kits from the data sheets of several suppliers, and adjusted a protocol by working with various concentrations and volumes of these ingredients. As a result, a fast and sensitive assay was applied as in the commercially available MAO kits with lower costs and clearer ingredients than those of the kits. The enzyme inhibition assay revealed that synthesized compounds have selective inhibition potency against hMAO-B. The compound 4e and 4f displayed IC50 values of 0.075 μM and 0.136 μM against hMAO-B, respectively. The reference drugs selegiline (IC50 = 0.040 μM) and rasagiline (IC50 = 0.066 μM) also displayed a significant inhibition against hMAO-B. The enzyme kinetic study was performed in order to observe the effect of the most active compound 4e on substrate-enzyme relationship and non-competitive inhibition of hMAO-B was determined. Cytotoxicity and genotoxicity studies were carried out and the compound 4e was found as non-cytotoxic and non-genotixic. Theoretical calculation of ADME properties suggested that compound 4e may have a good pharmacokinetic profile. The docking study of compound 4e revealed that there is a strong interaction between the active sites of hMAO-B and analyzed compound.