Purpose: To date, histological analysis is the gold-standard research tool for assessing cartilage integrity. Indentation features the benefits of assessing the main function of cartilage (shock absorption and force transmission) by its biomechanical parameters which could be altered by numerous factors. Furthermore significant cartilage metabolic disturbances can occur in cartilage in early stage of osteoarthrosis (OA) and prior to morphometric alterations. Therefore it could also be assumed that a normal macroscopic appearance of the cartilage does not ensure the absence of pathology. Despite the wide range of cartilage biomechanical testing methods in samples harvested from human or big animals’ models, rodents’ models which have an increasingly prominent role in OA research, are lacking of biomechanical assessment of their cartilage. Therefore, it is mandatory to develop a technique dedicated to the objective evaluation of the cartilage tissue quality. Methods: The original positioning of the femur allows to indent three different areas of interest of the cartilage surface corresponding to area physiologically exposed to load with various forces and orientation, various timing along the day and various frequency. Two different methods were used. Monotonic indentation was defined as a set of 5 successive indentations (controlled in force) within the region of interest. Each indentation was 200 μm distant from each other and were performed in the same sagittal plan of the medial condyle (at the center). Cyclic indentation was defined as a set of 5 successive cyclic indentations. All the 5 cyclic indentations were done with a quadratic load increment (from 0.05mN to 8 mN) resulting in 15 successive indentations at the same location repeated 5 times at 200 μm distant location. This allowed to investigate cartilage at different depth. In both type monotonic and cyclic indentation methods, elastic modulus (E), work of indentation (WI, area under the curve) and depth of indentation (h) were assessed. In the cyclic method they were assessed for each indentation force performed resulting in the determination of biomechanical material level properties at different depths. Distal femur were harvested from four 10 week-old female Lewis rats. Based on monotonic indentation, we first evaluated the reproducibility of the measurement by repositioning the indenter’s probe between each set of three monotonic indentations in the zone 1 of the medial condyle. Then, within each zone investigated and for each evaluated parameters, we determined in-vivo variability, defined by the average of the dispersion of the data between the 5 indentations which compose the monotonic indentation. In order to address the sensibility of the monotonic indentation, we assessed the difference between each investigated zone within the medial condyle (by t-test). Same investigations were also performed using cyclic indentation methodology within each zone of the medial condyle. Results: The mean reproducibility after probe repositioning was less than 10% for all evaluated parameters. In-vivo variability of all evaluated parameters within each investigated zone of interest was around 15% (range: 5-25%) in medial condyle as well as in lateral condyle. Despite this variability within each investigated zone we were able to determined distinct biomechanical properties relative to the zone investigated. In the medial condyle, in zone 1 as compared to zone 2 and 3, indentation depth was approximatively three times greater (p<.001) while WI and E were respectively four and two times greater (p<.05 and p<.001). Biomechanical properties were equivalent in zone 2 and 3. Based on the literature, this mapping of the cartilage biomechanical properties could correlate with in-vivo joint stress: since zone 1 is the most loaded zone during rat ambulation it could present the most elasticity. Using cyclic indentation method, at each investigated force inducing assessment of the hyaline cartilage thickness (without bone interaction), similar differences were observed as in the monotonic indentation method. This technic also succeeded to assess biomechanical deterioration of the cartilage in meniscectomized rats and in rats submitted to protein malnutrition (results presented at OARSI 2016 congress). Conclusions: These results indicate that the bioindentation of rat femoral cartilage is feasible, reproducible and enough sensible to detect biomechanical alterations between different zone of the cartilage as well as in biologic challenges.