Neuroblastoma (NB) is an aggressive pediatric malignancy originating from precursors of the sympathetic nervous system. As abnormal neural crest-derived sympathoadrenal cells are denoted by arrested differentiation, enforcing differentiation provides a promising therapeutic strategy. Currently, vitamin A-derived retinoic acids (RA) are used as a component of maintenance therapy. However, whether interactions exist with additional agents to improve differentiation effect, and enhance outcomes, remains unknown. Recent findings indicate that polyamine depletion via irreversible inhibition of ornithine decarboxylase (ODC) by difluoromethylornithine (DFMO) disrupts oncogenic translation and reprograms translation to enhance neural differentiation programs and repress proliferation programs. Whether transcriptional (RA-mediated) and translational (polyamine depletion-mediated) differentiation are synergistic is unknown. This study aimed to evaluate the effects of 13-cis-RA, DFMO, and their combination on SH-SY5Y cell differentiation and viability under amino acid-depleted conditions. 13-cis-RA effectively induced differentiation in arginine-depleted environments, which was amplified when combined with low doses of DFMO (0.5–1.0 mM). However, higher DFMO doses or combinations of DFMO and high RA induced significant cytotoxicity, reducing cell viability. These findings suggest a dose-dependent interplay, with potential synergy between DFMO and RA in promoting differentiation and altering cell viability. Findings indicate that the combination of RA and DFMO demonstrates potential as an enhanced differentiation therapy for neuroblastoma. These results provide a basis for further exploration of dose optimization and mechanistic studies to evaluate its therapeutic impact.