The absorptivities of polydiacetylenes (PDAs) used in Langmuir films or vesicles for the development of PDA sensor films or other applications such as nonlinear optics and field-effect transistors are not known, so the polymer contents cannot be deduced from experimental spectra. Here we introduce a novel method, using nuclear reaction analysis (NRA), that allows a quantitative determination of the polymer content X proportion of monomers that have been incorporated into PDA chains. We apply it to pentacosadiynoic acid (PCDA) evaporated microcrystalline films. A calibration curve giving X as a function of the area under an absorption spectrum normalized to the monomer areal density is obtained for blue and red PCDA. The method is applicable to all kinds of films and to other PDAs, provided films with known molecular areal density are available. An example of the application to a PCDA Langmuir film is given.