This study examined in vitro and in situ proteolytic characteristics of 8 lactobacilli strains isolated from Turkish Tulum cheese, consisting of L. plantarum, L. helveticus, L. rhamnosus, and L. paracasei, whose beneficial properties and safety concerns previously evaluated. The studied isolates presented versatile proteolytic activity with acidic, neutral, and alkaline proteases. The proteolytic activity of isolates against azocasein substrate was determined between 10.30 and 19.90 U/mg, and L. plantarum isolates had the highest proteolytic activity at varying pH values. Isolates 15, 449, 480 and 675 harboured proteinase genes in the catalytic region of prtR and prtP, and in the prtP/prtM intergenic region. All isolates were also evaluated in the production of fermented milk. As a result of SDS-PAGE analysis of fermented milk samples, L. plantarum isolates 449, 480 and 675 showed more proteolytic activity on milk caseins. Besides, isolate 480 was also effective in hydrolysis of beta-lactoglobulin. L. plantarum isolates resulted in an increase in amino acid (AA) concentrations especially in arginine, alanine, histidine, and proline during the 24-h fermentation of milk. AA production abilities varied depending on the strain. Generally, isolate 675 offered superior AA production ability. The proteolytic properties of cultures are effective on both human health and technological properties of the product in terms of reducing the allergenicity of milk proteins, forming functional peptides, and improving the sensory properties of the product. Based on the results of this study, L. plantarum isolates 449, 480 and 675 could potentially be used as starter cultures or co-cultures for these purposes.