Objective- The purpose of present study was to investigate the viability of equine fibroblast-like synoviocytes (FLSs) treated with doxycycline. Design- Experimental study. Sample population- FLSs from metacarpophalangeal joints of six skeletally mature horses. Methods- FLSs were established from synovial fluids of healthy joints. The cells were treated with different concentrations (1, 5, 10, 50, 100, 150, 300, 400 µg/ml) or without doxycycline for 48-hour. Viability of FLSs was determined by MTT assay and the Trypan blue dye exclusion method. Results- No significant differences were observed between viability of FLSs cultures treated with doxycycline until 150 µg/ml and control group (P>0.05). Doxycycline at 300 and 400 µg/ml significantly decreased FLSs viability (P<0.05). FLSs viability were 74.28% and 59.07% in 300 and 400 µg/ml, respectively, when measured by the MTT assay. Also FLSs viability at 300 and 400 µg/ml of doxycycline were 68.10% and 43.26%, respectively with Trypan blue exclusion method. Conclusion and Clinical Relevance- These findings demonstrate that doxycycline was not toxic for equine FLSs at concentration equal or less than 150 µg/ml in vitro. Further studies are needed to investigate the safety, efficacy and detrimental effects of doxycycline in equine joints.