Diffusion of tracer molecules in configurations of collagen fibrils may be used to determine anisotropy of fiber distributions in fluorescence microscopy experiments. Mathematical simulations are used to study the feasibility of these kinds of experiments. The anisotropic diffusion phenomenon can be modeled as a random walk process in simulated completely aligned fibers using the Monte Carlo method. We studied the relationships between the diffusion coefficients (either parallel or perpendicular to fiber orientation) and two influencing factors (density of fibers and relative size of fibers and tracer molecules). Using simulations and statistical analysis, we found that for a given fiber density, relatively bigger size tracer molecules are preferred in order to detect certain level of anisotropy of the fibers. If tracer molecules are too small compared with fibers, even high density of fibers can help little to detect any anisotropy.