Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. However, many proteins used for therapeutics and biosensors involve beta-sheet proteins such as antibodies. Accordingly, we explored the effect of several highly fluorinated amino acids on B-sheet stability including (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), (S)-5,5,5',5'-tetrafluoroleucine (Qfl), (S)-5,5,5,5',5',5'-hexafluoroleucine (Hfl), and (S)-pentafluorophenylalanine (Pff). Nine proteins based on the protein GB1 domain 16A T44A mutant (GB1) with various amino acids at the solvent exposed guest position 53 in the internal strand 4 were synthesized, purified, and investigated by thermal denaturation monitored by circular dichroism spectroscopy. Based on the thermal denaturation data, GB1 stability is affected by the amino acid at the guest position 53. Apparently, introducing fluorine results in more stable GB1 mutants (Pff > Phe, Hfl > Qfl > Leu, Atb > Abu). In particular, GB1 becomes more stable upon introducing fluorines by up to 0.35 kcal.mol(-1). residue(-1). Overall, these results suggest that fluoro-amino acids may be worthwhile building blocks to explore for stabilizing B-sheet proteins, which are especially important for biotechnologies such as protein therapeutics and biosensors.