Glycine receptors (GlyRs) mediate inhibitory neurotransmission within the spinal cord and play a crucial role in nociceptive signalling. This makes them primary targets for the development of novel chronic pain therapies. Endogenous lipids have previously been shown to modulate glycine receptors and produce analgesia in pain models, however little is known about what chemical features mediate these effects. In this study, we characterised lipid modulation of GlyRs by screening a library of N-acyl amino acids across all receptor subtypes and determined chemical features crucial for their activity. Acyl-glycine's with a C18 carbon tail were found to produce the greatest potentiation, and require a cis double bond within the central region of the carbon tail (ω6 – ω9) to be active. At 1 µM, C18 ω6,9 glycine potentiated glycine induced currents in α and αβ receptors by over 50%, and α, α, αβ and αβ receptors by over 100%. C18 ω9 glycine (N-oleoyl glycine) significantly enhance glycine induced peak currents and cause a dose-dependent shift in the glycine concentration response. In the presence of 3 µM C18 ω9 glycine, the EC of glycine at the α receptor was reduced from 17 µM to 10 µM. This study has identified several acyl-amino acids which are positive allosteric modulators of GlyRs and make promising lead compounds for the development of novel chronic pain therapies.