Developing more effective targeting of chemotherapeutics is currently one of the most pressing needs in oncology. One promising approach is to utilize the acidic microenvironment of cancer tumors, which is a universal property of all cancers. pH (Low) Insertion Peptide (pHLIP) is a soluble peptide that binds and inserts into cell membranes under acidic conditions. However, developing pHLIP into a novel cancer targeting agent will require informed design of a variant that can distinguish between the pH range of 7.2 (healthy tissue) and 6.8 (most cancers). One of the key steps in pHLIP function is the process of folding into an α-helix before insertion. Partial helical formation of pHLIP under alkaline conditions has been reported in literature, and recent results from our group [1] support this claim. These observations, coupled with biophysical theory of peptide folding [2], leads to our hypothesis that the apparent pKa of insertion of pHLIP is tied to its helix-forming propensity in solution. We have tested this hypothesis through long timescale (∼μs) molecular dynamics simulations of pHLIP in implicit solvent. Comparison with the insertion behavior observed in two variants of pHLIP that use non-natural amino acids [3] allows us to tie the unique properties of pHLIP to its primary amino acid sequence. Application of the Lifson-Roig model of helix-coil transition to our results yielded a complete thermodynamic description of state I of pHLIP. Collectively, our results are the first steps towards informed design of pHLIP variants with properties ideally suited to target cancer cells.[1] Renet. al. Proteins., (Submitted);[2] Almeida et al., BBA, 1818:178 (2012);[3] Onyango et al., Angew. Chem. Intl. Ed., 54:3658 (2015).