Targeting bromodomain-containing protein 9 (BRD9) represents a promising strategy for the development of new agents endowed with anticancer properties. With this aim, a set of 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based compounds was investigated following a combined approach that relied on in silico studies, chemical synthesis, biophysical and biological evaluation of the most promising items. The protocol was initially based on molecular docking experiments, accounting a library of 1896 potentially synthesizable items tested in silico against the bromodomain of BRD9. A first set of 21 compounds (1-21) was selected and the binding on BDR9 was assessed through AlphaScreen assays. The obtained results disclosed compounds 17 and 20 able to bind BRD9 in the submicromolar range (IC50 = 0.35 ± 0.18 μM and IC50 = 0.14 ± 0.03 μM, respectively) showing a promising selectivity profile when tested against further nine bromodomains. Taking advantage of 3D structure-based pharmacophore models, additional 10 derivatives were selected in silico for the synthetic step and binding assessment, highlighting seven compounds (22, 23, 25, 26, 28, 29, 31) able to selectively bind BRD9 among different bromodomains. The ability of the identified BRD9 binders to cross artificial membranes in vitro was also assessed, revealing a very good passive permeability profile. Preliminary studies were carried out on a panel of healthy and cancer human cell lines to explore the biological behavior of the selected compounds, disclosing a moderate activity and significant selectivity profile towards leukaemia cells. These results highlighted the applicability of the reported multidisciplinary approach for accelerating the selection of promising items and for driving the chemical synthesis of novel selective BRD9 binders. Moreover, the low molecular weight of the reported 2,4,5-trisubstituted-2,4-dihydro-3H-1,2,4-triazol-3-one-based BRD9 binders suggests the possibility for further exploring the chemical space in order to obtain new analogues with improved potency.