In this thesis, a method for studying lateral mobility of integrin α5β1 on a single-molecule level was set up and applied. Integrins are transmembrane, heterodimeric glycoproteins, mainly responsible for the processes of cell anchoring, adhesion, and migration. Lateral diffusion of integrins on the membrane is an important aspect of the cell adhesion and mobility, but how it affects the cell’s biochemical and biomechanical processes is still not thoroughly investigated. Quantifying movement of membrane proteins and elucidating their biological function has greatly been improved with Single-Particle Tracking (SPT). SPT is based on the sparse labeling of the target molecule, recording the movement of particles in a series of videos by optical microscopy and analysis of videos by image-processing algorithms that locate individual molecules with nanometric precision and establish a correspondence between particles in each frame. Understanding integrin’s complex behavior at the single-molecule level could potentially lead to the development of treatments for conditions linked to integrindysfunction such as injury or cancer. The objective of this thesis was to implement and optimize the SPT method for studying the diffusion of α5β1 integrin at the single-molecule level. Diffusion of integrins labeled with the reduced half-antibody fragment was compared to those labeled with the whole antibody to test if the immuno-labeling is influencing integrin movement through dimerization and artificial clustering. A statistically significant difference in the distribution of two types of labeled integrins was determined.