The reactivity ratios in a copolymerization are needed to predict the microstructure (random, gradient, block or alternating) of the produced copolymer. This microstructure reflects on the physical properties of the polymeric material. Conventional ways to determine these reactivity ratios demand in most cases tedious laboratory work and several experiments at different monomer feed compositions. Here, a novel method is described to derive these ratios from a single MALDI-ToF-MS spectrum obtained at one feed composition by employing either a Monte Carlo approach to numerically simulate a first order Markov chain or the analytical form of the first order Markov chain. A single MALDI-ToF-MS spectrum proved to give very good estimates of the reactivity ratios of comonomers from copolymer's synthesized by free radical polymerization, ring-opening polymerization of lactones and lactides, or ring-opening copolymerization of anhydrides plus epoxides.