Interfiber friction in paper exists in fiber suspensions, fiber flocs, and fiber networks. The interfiber friction force is, therefore, important both in papermaking and in the use of paper. The objective of this research is to develop a methodology using atomic force microscopy (AFM) for the direct measurement of the friction force between pulp fibers. Different factors such as AFM scanning velocity, contact area, and fiber surface roughness were investigated. The results show that AFM is an effective tool for measuring micro-scale interfiber friction forces. Both AFM scanning velocity and fiber surface roughness affect the measured results. The coefficient of friction increases, but the initial adhesion force decreases, with increasing fiber surface roughness.