The study was aimed to compare the effect of chloroquine (CQ) on the expression of apoptosis and autophagy genes in the two tumor cell lines, leukemia MOLT-3 and neuroblastoma IMR-32, cultured in parallel in complete growth and serum-free RPMI-1640 or DMEM media, respectively, for 24 and 48 h. Cell viability was assessed by the MTT method, gene expression—by real-time PCR. For MTT assay, the cells were incubated with 10–100 µM CQ. The expression of apoptosis (CASP3, BAX, BCL2) and autophagy (ULK1, BECN1, MAP1LC3B) genes was studied using 30 and 50 µM CQ, which exerted a considerable inhibitory effect on the viability of cell of both lines, but did not cause their complete death. The sensitivity of both cell lines to CQ was higher in the serum-free medium, however, the expression of apoptosis and autophagy genes substantially differed between them. In MOLT-3 cells, mRNA levels of the pro-apoptotic genes CASP3 and BAX increased after 24-h incubation in the serum-free medium, whereas in IMR-32 cells, the expression of these genes increased only after 48-h incubation in the presence of higher CQ concentration. In the cells of both lines, 24-h CQ treatment resulted in enhanced expression of the anti-apoptotic gene BCL2. Under conditions of nutrient deficiency, the genes responsible for the three stages of autophagy (ULK1, BECN1, MAP1LC3B) demonstrated different combinations of their stimulation in MOLT-3 cells, but none of the treatment protocols influenced ULK1 and MAP1LC3B gene expression in IMR-32 cells. Overall, a 24-h treatment with CQ under conditions of serum starvation is optimal for autophagy modulation in MOLT-3 cells. In IMR-32 cells, CQ exerts no considerable effect on the expression of autophagy genes, and their decreased viability appears to be due to activation of other mechanisms.