The interactions that occur at the cell membrane surface are essential starting points for understanding signal transduction pathways. Ras/RAF/MEK/ERK is part of the mitogenactivated protein kinase (MAPK) signaling pathway that is frequently known to harbor an oncogenic mutation. Activation of downstream effector RAF is a result of recruitment to the plasma membrane via membrane-localized Ras. However, an incomplete quantitative understanding of how these multivalent molecular assemblies behave at the cell surface and their mechanistic binding events that modulate downstream signaling remains debated. Here, we use Ras and RAF proteins as a model system to quantitively study their interactions on a supported lipid bilayer. In this work, we (1) demonstrate an in-vitro reconstitution assay that measures interactions between lipids and membrane-anchored Ras, and (2) to experimentally test if anionic lipids modulate the accessibility to effector binding sites of Ras. By combining single-molecule imaging and spectroscopy techniques on supported lipid bilayers, we can quantitatively study the binding interactions at the membrane surface. Through this approach, we found that the accessibility of Ras to the Ras-binding domain (RBD) of RAF remains the same on anionic lipid membranes such as PS and PIP2. Overall, our results and future work will provide a better approach to studying lipid dynamics and multivalent assembly mechanisms involved in membrane signaling.