Background and Purpose: Osteoarthritis, a major cause of disability in developed countries does not have effective treatment. Activation of TLR4 and innate immune response factors contribute to osteoarthritis progressive cartilage degradation. There are no clinically available TLR4 inhibitors. Interestingly, the antidepressant amitriptyline could block this receptor. Thus, we evaluated amitriptyline anti-TLR4 effects on human osteoarthritis chondrocytes in order to repurpose it as an inhibitor of innate immune response in joint inflammatory pathologies.Experimental Approach: Using in silico docking analysis, RT-PCR, siRNA, ELISA, proteomics and clinical data mining of drug consumption, we explored the clinical relevance of amitriptyline blockade of TLR4-mediated innate immune responses in human osteoarthritis chondrocytes.Key Results: Amitriptyline bound TLR4 but not IL-1 receptor. Interestingly, amitriptyline binding to TLR4 inhibited TLR4-and IL-1 receptor-mediated innate immune responses in human osteoarthritis chondrocytes, synoviocytes and osteoblasts cells.Amitriptyline reduced basal innate immune responses and promoted anabolic effects in human osteoarthritis chondrocytes. Supporting its anti-innate immune response effects, amitriptyline down-regulated basal and induced expression of NLRP3, an inflammasome member from IL-1 receptor signalling linked to osteoarthritis and gout pathologies. Accordingly, mining of dissociated and aggregated drug consumption Abbreviations: 2-ddCt, 2-delta-delta cycle threshold; dCt, delta CT; DDA, data-dependent acquisition; NAMPT, nicotinamide phosphoribosyl transferase; SWATH, sequential window acquisition of all theoretical mass spectra.