Microalgae are fast growing organisms that can be used as feedstock for the production of biofuels. The metabolism of microalgae can be manipulated by exposing them to different environmental conditions for favoring the accumulation of lipids, carbohydrates or proteins. For example, a change in growth conditions can cause the accumulation of large amounts of lipids, representing an opportunity for biodiesel production. Monitoring changes in the composition of microalgal cells is therefore important in assessing new growth conditions. However, at present, most techniques are time consuming, invasive and expensive. Here we have used FTIR microscopy to quantify lipid, protein, and starch accumulation in Neochloris minuta cells grown in the presence and absence of nitrogen. Under nitrogen deprivation the cellular lipid composition increases by a factor of 2.4, the cellular protein concentration decreases by ~60% while the starch concentration is unaltered. These estimates of biochemical composition were derived using a variety of analytical methods, and form the basis for establishing to what extent FTIR microscopy can be used as a probe of cellular biochemical composition. We find that the distribution of materials in Neochloris minuta cells estimated directly from the FTIR spectra compare favorably with that estimated using these other analytical methods. FTIR spectroscopy is shown to be a versatile and easy-to-use tool for estimating distributions of biological materials in microalgal cells.