We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.