AbstractAccurate estimation of the nonaqueous phase liquids (NAPLs) saturation such as chlorinated organic compounds (COCs) in aquifers is crucial for the proper remediation of contaminated groundwater. A combination of conservative and partitioning tracers (PTs) are commonly used to assess NAPL saturations in the subsurface at COC release sites, using the partitioning interwell tracer test (PITT). In this study, five fluorescent dyes were assessed as PTs to estimate the saturation of octanol and 3 COC NAPLs in soil columns. PT experiments required an initial assessment of both partitioning (NAPL/water and octanol/water) and linear free‐energy relations. The predictability of the partition coefficients was correlated to the pH of the two‐phase fluids for both systems (NAPL/water and octanol/water). The COC NAPLs were acidic and some PTs with acid‐base properties, like fluorescein, are easily influenced by pH. The PITT experiments were performed in a column packed with glass beads, using rhodamine WT as PT because of its particular specificity for the complex mixture of NAPLs and sodium chloride as the inert tracer. Breakthrough curves of rhodamine WT were examined to estimate the saturation of a NAPL made of a complex mixture of COCs. The DNAPL residual saturation estimation accuracy was sensitive to both pH variations and the water velocity. The latter was represented by an exponential function which resulted from non‐equilibrium measurements, heterogeneous sweeping of the contaminated sample, and redistribution of the NAPL droplets in the medium.