Elucidating the chemical composition of microfluidic flows is crucial in both understanding and optimising reactive processes within small-volume environments. Herein we report the implementation of a novel detection methodology based on Attenuated Total Reflection (ATR)–Fourier Transform Infra-Red (FTIR) spectroscopic imaging using an infrared focal plane array detector for microfluidic applications. The method is based on the combination of an inverted prism-shape ATR crystal with a poly(dimethylsiloxane)-based microfluidic mixing device. To demonstrate the efficacy of this approach, we report the direct measurement and imaging of the mixing of two liquids of different viscosities and the imaging and mixing of H2O and D2O with consecutive H/D isotope exchange. This chemically specific imaging approach allows direct analysis of fluid composition as a function of spatial position without the use of added labels or dyes, and can be used to study many processes in microfluidics ranging from reactions to separations.