We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na+, K+, Ca+2, Mg+2), and the initial extra-liposomal amount of luciferin Symbol on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and Symbol was established, and under our conditions the maximum LC was attained using an Symbol of around 2.6 μmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between Symbol and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1 × 10−8 cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.