Osteoarthritis (OA) is the most common major disabling disease in humans and horses. Hyaluronic acid (HA), naturally abundantly present in synovial fluid (SF), is thought to have crucial impact on the functional rheological and biochemical features of SF in healthy and osteoarthritic joints. Here we present comparative measurements of HA concentration in SF from 35 normal and osteoarthritic equine joints, between two different approaches. On the one hand, an established biochemical HA-specific Enzyme–Linked Immunosorbent Assay (ELISA) assay was employed, which determined that SF in healthy and osteoarthritic equine joints is characterized by HA concentration of ca 0.3–2 mg/mL and 0.1–0.7 mg/mL respectively. On the other hand the same SF samples were also examined with a new exploratory approach of finding out HA concentration, which is based on SF rheology. This was done following “calibration” using appropriate model HA solutions. Comparative analysis of the results obtained by both the biochemical and the rheological approaches, revealed that in most cases the rheological approach greatly overestimates HA concentration in SF, by ca 3 to 8 times and 6 to 11 times, in healthy and diseased SF respectively. Overall these findings support the notion that, contrary to the established view, HA may not be the major contributor of equine SF rheology. This should be taken into account for the development of new more effective preventive strategies, as well as more effective early-stage interventions in osteoarthritis.