A microsphere-surface adhesion characterization technique based on the rolling resistance moment of the adhesion bond is reported and demonstrated. With a nanomanipulation system, an increasing lateral pushing force is applied to a polymer microsphere adhered on a silicon substrate in the ambient environment, and the rolling displacement of the particle in response to the external force is recorded. The work of adhesion for polymer microspheres to the silicon substrate is extracted from the force-displacement curves. Unlike the traditional colloid probe technique where particles are glued to the cantilever beam and the normal detachment force as a function of the out-of-plane displacement is measured, in the current work the work of adhesion is directly determined from the measured rolling resistance moment and the proposed approach requires no prior knowledge of the particle diameter. In addition to the work of adhesion for the set of microspheres, the critical angles of rolling prior to rolling are estimated. The reported results form further experimental evidence for the existence of a rolling resistance moment.