The aim of this study was to compare the efficiency of two RNA isolation methods for subsequent quantitative gene expression analysis in primary cultures of rat hippocampal neurons: a modified protocol using TRIzol™ reagent and the column-based GeneJET™ RNA Purification Kit. It was established that both methods provide similar RNA concentration and purity, as well as comparable qPCR reproducibility. No significant differences were observed between the methods in terms of A260/280 ratio, RNA concentration, or amplification variability (p > 0.05). Thus, the optimized TRIzol™ protocol demonstrated an RNA purification quality close to that of the column-based method and can be considered a reliable and cost-effective alternative for molecular biology studies performed on cultures with a limited amount of biological material.