Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

►Membrane-inserted peptides do not affect the lo to ld spectral shift of laurdan. ►Membrane-inserted peptides do not affect the lo to ld spectral shift of di-4-ANEPPDHQ. ►Neither α-helical nor β-structure peptides affected the spectra of laurdan. Neither α-helical nor β-structure peptides affected the spectra of di-4-ANEPPDHQ. ►LUVs retained their integrity at high concentrations of membrane inserted peptides. ►Laurdan and di-4-ANEPPDHQ are appropriate for assessing lipid order in cells.