Methods for the quantitative determination of magnetic nanoparticles in biological matrices, in the frame of biomedical applications, are required to evaluate the particles biodistribution after systemic administration. AC magnetic susceptibility measurements are an alternative method to quantify magnetic nanoparticles in tissues, being able to provide also information on the particle transformations over time and allowing the distinction of the particles from other endogenous species such as the ferritin iron cores. The protocol for particle quantification using AC magnetic susceptibility measurements is described in detail in this article. A summary of synthesis routes towards magnetic nanoparticles is also provided.