Lubrication is an important function of many tissues of the body, including the articular cartilage, pericardium, pleural membrane, and intestinal lining. The molecular mechanisms governing friction and lubrication in vivo are poorly understood in most cases, largely due to a lack of appropriate testing methodologies. Here we present an approach in which friction was measured in situ on murine cartilage by colloidal probe microscopy. Sliding occurred between a chemically functionalized microsphere and the cartilage surface of the murine femoral head. Hydrostatic and hydrodynamic lubrication, both of which contribute to cartilage lubrication under some conditions, were minimized in this testing configuration, allowing a focused study of boundary lubrication. Other properties relevant to friction were measured in the same configuration. Surface roughness was measured by raster scanning the probe across the surface, compressive stiffness was measured by indenting the cartilage surface with the probe and fitting the approach portion of the curve to the Hertz model, and adhesion was measured from the retract portion of the force curve. We assessed the effect of each of these factors, as well as sliding distance and sliding speed, on friction and concluded that interfacial shear was the principal mechanism of shear generation in this system. Little to no friction resulted from plowing forces, collision forces, or energy losses during normal deformation. Friction coefficients measured on murine cartilage (0.25±0.11) were similar to coefficients measured on porcine cartilage (0.23±0.09) and were in general agreement with previous measurements of boundary friction on cartilage by other researchers. This technique can be applied to future in situ measurements of friction and mechanical properties on genetically modified small animals and soft surfaces in general.