The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP® peptide (Short pHLIP®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP® peptide spans lipid bilayer of membrane similar to Long pHLIP® peptides. Truncated pHLIP® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs.