On the nano- and micro-scale, conventional liquid-based lubrication cannot be utilized to minimize friction due to excessive surface tension and related effects. To overcome this limitation, solid lubricants suitable for use in nano- and microscale systems are needed. Being a two-dimensional material with outstanding mechanical properties, graphene emerges as a promising candidate for this purpose. Motivated as above, this M.S. thesis presents a comprehensive investigation of the nanotribological properties of mechanically-exfoliated graphene conducted via atomic force microscopy (AFM), whereby special emphasis is placed on the effect of interface structure. Graphene samples ranging from single- to few-layers were fabricated using the mechanical exfoliation method and transferred onto Si/SiO2 substrates. By utilizing optical microscopy and Raman spectroscopy, graphene akes exhibiting single- and bi-layer regions were located and identified. Furthermore, using topographical maps and associated profiles obtained via AFM, 3-, 4-layer and bulk graphite regions were found. Moreover, AFM probes were calibrated both for accurate normal force readings, and for obtaining quantitative friction force data from lateral force measurements conducted via contact-mode AFM under ambient conditions. Following sample preparation, identification and probe calibration, experiments aimed at measuring the effect of applied load on friction of single- and 2-, 3-, 4-layers of graphene were performed, confirming previous results reported in the literature as explained by the puckering phenomenon. Additionally, the effect of tip radius and thus, contact area, on the frictional behavior of graphene was quantitatively measured. In particular, thermal evaporation- and PECS (precision etching coating system)-based coating of gold onto AFM probes were utilized to modify tip radii. Results led to the determination of a new parameter affecting friction on graphene: interface roughness. In collaboration with scientists from UC Merced who performed molecular dynamics simulations complementing the experiments presented here, the effect of substrate roughness, which may be in addition to, or dominant over, the puckering phenomenon, was analyzed in terms of the frictional behavior of graphene. Presented experimental results provide a new perspective towards the layer-dependent frictional behavior of graphene, underlining the in uence of substrate roughness in addition to the phenomenon of puckering that is well-studied in the literature.