Quantifying the rate and the amount of drug entering live cells is an essential part of the medicine development process. Infrared spectroscopy is a label-free, chemically selective tool for analyzing the composition of live cells in culture that has the potential to quantify, in situ, the amount of drug entering living cells in a nondestructive manner, although its sensitivity is currently limited. This paper is the first to demonstrate in situ quantification of the cancer drug, fluorouracil, in live cells at a therapeutically relevant concentration using Fourier transform infrared spectroscopy. To achieve the required improvement in detection and quantitation limits of the IR measurement, two strategies were exploited. First, a sampling method called multibounce attenuated total reflection was used to optimize the signal while second, a long pass filter in combination with a mercury cadmium telluride detector was used to reduce the instrument noise. Using these novel adaptations, it was possible to quantify 20 μM of fluorouracil in cell culture medium using a standard FTIR instrument, while it was possible to quantify and measure the flux of fluorouracil in situ in living cells treated with an 80 μM drug.