Diatoms are the most successful biomass producers worldwide. Therefore, physiological and chemical methods to measure the cell response to a variety of abiotic factors are the focus of recent research. We used the two model diatoms Cyclotella meneghiniana and Skeletonema costatum for the development of Fourier transform infrared (FTIR) spectroscopy-based methods to measure simultaneously the elemental composition of the cells and their cell-specific physiological properties. The cells were grown in chemostat cultures to study the response of Si limitation. The model organisms showed different reactions in terms of their cell properties. Si limitation was accompanied by a drop in the growth rate, a reduced content in Si per cell and a decreased Si : C ratio. Furthermore, the C allocation pattern was changed in both diatoms under Si limitation, as shown by FTIR spectroscopy. Moreover, we used FTIR spectra to develop PLS (partial least square) regression methods to predict the Si content and the Si : C ratio for single as well as multiple species. All PLS regression models were validated by standard chemical methods and showed good prediction accuracy, with the coefficient of determination R(2) being ≥0.93. We could show that it is possible to monitor phytoplankton properties such as C allocation, the Si content and the Si : C ratio at the same time via FTIR spectroscopy.