Fructo-oligosaccharides (FOS) are mixtures of oligosaccharides composed of fructose and glucose units. As their composition is determined by the synthesis conditions, the goals of this work were: (a) to engineer FOS of different composition by adjusting the sucrose concentration used as initial substrate; (b) to define partial least square (PLS) based-models to quantify all the sugars present in the reaction medium directly from the FTIR spectra. The yield of each reaction was calculated as the percentage of initial sucrose converted to each oligosaccharide, as monitored by HPLC. In parallel, the reactions were followed by FTIR. Six different PLS models aiming to determine the concentration of each carbohydrate present in the reaction medium were calibrated and independently validated. The means of predicted values fitted well to those obtained by HPLC. Determining FOS composition directly from the FTIR spectra represents a useful tool to monitor enzymatic synthesis, with strong impact at both an academic and an industrial level.