Endometriosis is the growth of endometrial tissue outside of the uterine cavity. Its aetiology remains obscure, and it is difficult to diagnose ranging from asymptomatic to debilitating disease. Mid-infrared (IR) spectroscopy has become recognised as a potential clinical diagnostic tool. Biomolecules absorb mid-IR (4000 cm−1 to 400 cm−1) and from this, a biochemical-cell fingerprint in the form of an absorbance spectrum can be derived. We set out to determine if IR spectroscopy could be used to identify underlying biochemical differences between endometrial tissues growing outside of the uterus (ectopic) from endometrial tissue of the uterus (eutopic). For comparative purposes, endometrial tissues from endometriosis-free women were also obtained (benign eutopic). Attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy or transmission FTIR microspectroscopy was employed for spectral acquisition. Principal component analysis (PCA)-linear discriminant analysis (LDA) was used for chemometric analysis. A clear segregation was exhibited between the three categories independent of inter-individual confounding differences. Importantly, there was a marked difference between eutopic endometrial tissue from patients with or without endometriosis. This indicates that IR spectroscopy coupled with multivariate analysis (e.g., PCA-LDA) may provide a non-invasive diagnostic tool for endometriosis. By analysing the underlying biochemistry of these endometrial tissues, this approach may facilitate a better understanding of this pathology.