AbstractTrifluoroacetate (CF3COO−, or TFA) is almost always present in commercially synthesized peptides. Unfortunately, it has a strong infrared (IR) absorption band at 1673 cm−1, significantly overlapping or even completely obscuring the amide I band of a peptide. In such cases TFA must be removed from the solution in order to be able to use IR absorption spectroscopy for peptide secondary structure determination. The most convenient and widely used procedure involves peptide lyophilization from a 0.1 M HCl solution. In our studies of the tryptophan‐rich antimicrobial peptide indolicidin, we have found that caution should be taken when using this HCl concentration. High HCl concentrations (>10 mM in unbuffered solutions and>50 mM in buffered solutions) may modify the peptide structure and reduce its thermal stability, thereby interfering with subsequent structural investigations of the peptide. Our results indicate that HCl concentrations between 2 and 10 mM are adequate to remove essentially all TFA impurities without any modification of the peptide secondary structure. Copyright © 2006 European Peptide Society and John Wiley&Son