Labelling of bacterial membranes using exogenous fatty acids has proven to be a valuable tool to investigate molecular interactions by in-cell solid-state nuclear magnetic resonance (ssNMR) spectroscopy, notably with antimicrobial peptides. However, the mechanism by which this labelling takes place in non-mutated bacteria has not yet been investigated. In this work, we propose a rapid method to assess the fate of the fatty acids during the labelling of bacteria, involving two different methylation schemes and gas chromatography coupled to mass spectrometry. We applied this approach to Gram(+) and Gram(−) bacteria grown with deuterated palmitic acid under different conditions. We assessed the extent of labelling, then the resulting membrane rigidity by 2H ssNMR. Our results reveal that the labelling mechanism depends on the detergent used to micellize the fatty acids. This labelling can be either active or passive, whether the fatty acids are metabolized and used in the phospholipids biosynthesis, or remain unmodified in the membrane. We discuss the best labelling protocol for studying peptide-membrane interactions.