Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was evaluated for both qualitative and quantitative analysis of insulin entrapped within poly(D,L‐lactic‐co‐glycolic acid) nanoparticles. Quantitation was performed by adding an internal standard (arg‐insulin) to defined and unknown sample solutions, in order to reduce point‐to‐point and sample‐to‐sample variability. The ratio of the peak height of insulin to the peak height of arg‐insulin was plotted against the insulin concentration. In this way, an excellent linear relationship was found (R2 > 0.99). This method of quantitation was compared with classical UV spectroscopy and reverse‐phase high‐performance liquid chromatography measurements. All methods provided close final drug loading values for the insulin‐loaded nanoparticle batches tested. Additionally, with respect to molecular stability, covalent insulin dimers were found only at trace levels in those nanoparticles. Compared with other methods, MALDI‐TOF MS is a valuable tool for the characterization of proteins from nanoparticles, because no extensive extraction and complex sampling procedures are required.