The pathological hallmark of a range of neurodegenerative diseases, including Motor Neurone Disease (MND) is the presence of protein inclusions and aggregates in affected neurons. Aggregates progress continually from the site of onset to disturb cellular processes, ultimately resulting in neuron degeneration. The only intracellular degradative pathway that can purge the cells of these misfolded proteins, aggregates, and dysfunctional organelles is autophagy. Since impaired/reduced autophagy may contribute to MND pathogenesis; upregulating autophagy offers a potential therapeutic option. This study aimed to develop and synthesize novel pharmacological agents that have non-toxic autophagy-inducing properties in motor neuronal NSC-34 cells. A series of peptides analogues of the evolutionary conserved domain of Beclin1 protein, the master regulator of autophagy, were designed and synthesized. In order to enhance the cellular uptake and increase the cytosolic bioavailability, the peptides were cyclized through disulfide, thioether and lactam bond formation, and conjugated to a cell-penetrating peptide domain (a fragment of apolipoprotein E (ApoE)), and an endosomal escape domain, hemagglutinin-2 protein (HA2). The autophagy-inducing effect of the peptides was determined by measuring the level of autophagosome-associated LC3-II, a marker of macroautophagy, in NSC-34 cells. We have shown that the peptides are capable of inducing autophagy in NSC-34 cells at 5 micromolar concentration, which is 10-fold more potent than the previously reported Beclin-1 analogues. We have also shown that cyclization and addition of a cell-penetrating peptide and endosomal escape domain to the Beclin peptides further increases their autophagy-inducing potency. More importantly, the most potent peptide analogue, HA2-ApoE-Beclin-3, significantly decreased the severe and aggregate-prone form of mutant SOD1 (A4V) protein level. This study provides in vitro evidence for a novel autophagy-inducing approach that could have a potential therapeutic benefit in MND and other proteinopathies.