Lipophilicity is one of the most important physicochemical parameters to control during early-stage drug development. Optimization of lipophilicity is critical especially for larger compounds, as the margin for achieving both acceptable passive cell permeability and aqueous solubility thins tremendously as molecular weight increases. Larger drug leads have recently become more highly pursued due to the advent of encoded library technologies (DEL, mRNA display) and for their unique ability to bind to extended protein surfaces and disrupt protein-protein interactions. To ensure that early-stage lead compounds beyond the rule-of-5 are optimized to simultaneously exhibit acceptable aqueous solubility and passive cell permeability, a rapid and accurate empirical measurement of lipophilicity is desirable. The gold standard of lipophilicity is shake-flask partition coefficients, which are prone to sensitivity issues and experimental error. However, chromatographic assessments of lipophilicity which reflect hydrocarbon/water partition coefficients have significant potential to rapidly assess passive cell permeability and aqueous solubility profiles for early-stage peptide macrocycles in development. By applying the combinatorial and encoding power of DNA-encoded libraries, chromatographic measurements of lipophilicity could rapidly translate DEL hits into lead compounds by selecting compounds with the highest potential for passive cell permeability and aqueous solubility. Chapter 1 is an investigation of the potential to use chromatographic measurements of lipophilicity to reproduce 1,9-decadiene/water partition coefficients and predict passive cell permeability for complex, mass-encoded libraries of peptide macrocycles. We constructed a non-linear regression model from over 200 peptide macrocycles which can estimate 1,9-decadiene/water partition coefficients (Log Ddd/w) from a single capacity factor measurement. This model predicts Log Ddd/w values with high accuracy for a diverse set of peptide macrocycles. These estimated values, called Log EDdd/w, can be used to determine lipophilic permeability efficiency (cLPE), a quantitative metric that is descriptive of permeability-relevant lipophilicity and is directly related to the amount of exposed hydrogen bond donors. Log EDdd/w correlates strongly with PAMPA and MDCK permeability for peptide macrocycles in the soluble regime. This chromatographic lipophilicity assay is sensitive to minute structural differences, such as inversion of a single stereocenter, and MDCK simulations demonstrate that it can capture desirable conformational features for peptide macrocycles, such as intramolecular hydrogen bonding. In Chapter 2, chromatographic conditions for assessing individual compound purity and measuring permeability-relevant lipophilicity were developed for DNA-encoded libraries (DELs). Optimized conditions were developed on model compounds, featuring a series of hexapeptide diastereomers conjugated to 48bp DNA whose on-DNA capacity factors reflect their off-DNA CACO-2 cell permeabilities. A 62,581 member DNA-encoded library of peptide macrocycles was synthesized which features significant structural diversity in the peptide backbone. A novel method was developed to partition the library chromatographically and sequence the collected library fractions to produce sequencing count chromatograms which can be used to assess purity and calculate retention times for individual library members. Additionally, an analytical framework was developed utilizing encoded null truncations to identify synthetic truncations from the library synthesis and determine the relative purity of each DNA-encoded library member. The on-DNA retention times from the sequencing count chromatograms strongly correlated with off-DNA retention times and PAMPA permeability for 32 resynthesized library members spanning four macrocyclic ring sizes. This novel strategy is an ultra-high throughput method to profile reactivity in DNA-encoded libraries. Furthermore, it provides purity data about each individual encoded library member and measures permeability-relevant lipophilicity at an unprecedented scale. This chromatographic method can be applied to inform the design of future DELs or used to guide hit resynthesis efforts from DEL protein selection experiments.