Atomic force microscopy (AFM) offers an exciting tool for quantitatively investigating the physiological activities at single-cell and single-molecule levels. Here, we describe the applications of AFM single-molecule force spectroscopy (SMFS) in investigating the molecular specific interactions on the cell surface. This chapter begins with an introduction to the rituximab (an anti-CD20 antibody)–targeted therapy of lymphoma and discusses the challenges it faces. The following section presents a detailed description of SMFS, an important technique that enables AFM to locate and manipulate single molecules on the cell surface. Next, it presents how to measure the interaction forces between single CD20–rituximab pair directly on the tumor cells prepared from clinical lymphoma patients based on fluorescence recognition. Next, it presents how to map the nanoscale distribution of CD20 antigen on the tumor cells and analyze its potential role in the clinical treatments of rituximab. The nanoscale investigations of CD20–rituximab interactions on the surface of tumor cells using SMFS will be clinically useful for us to address the problem of resistance in the rituximab therapy of lymphomas.