Autophagy, referring to macroautophagy, is an evolutionarily conserved degradation pathway. Through autophagy cells can degrade damaged organelles, lipid vesicles and misfolded protein aggregates with implications in various pathological conditions, including neurodegenerative diseases, cancers, and infectious diseases. Autophagy is one of the major intracellular membrane-trafficking processes and its morphology includes the initiation, maturation, transportation and degradation of autophagosomes, where the double-membrane autophagosomes package the cargo for degradation. Therefore, understanding how autophagosomes form and are regulated is important in this field. Here we conducted a genome-wide siRNA screen using a high-throughput imaging system to identify undiscovered regulators in autophagosome formation. In this study, HEK293 cells stably expressing GFP-DFCP1 (GFP-tagged zinc finger FYVE-type containing 1) were used and amino acid starvation was used to induce autophagy. After the first round of primary screening, a small-scale screen was conducted with the same conditions including 384 candidates and additionally with these candidates HEK293 cells stably expressing GFPLC3 (GFP-tagged microtubule associated protein 1 light chain 3) were used to monitor the late steps of the autophagy process. From these rounds of screening, 39 candidates were selected and validated by investigating early autophagosome markers for their effects on autophagosome formation. Finally, five of the best candidates were confirmed based on their depletion effects on autophagy. Among these five candidates, DCAKD (dephospho-CoA kinase domain containing), WDR91 (WD repeat domain 91) and WDR65 were further investigated. Preliminary data using both RNAi and CRISPR Cas9 showed that DCAKD affected the accumulation of some early autophagy markers on initiation membranes in autophagosome formation. Interestingly, protein levels of canonical autophagy markers remained unchanged in DCAKD-deficient cells. Another candidate WDR91 showed the ability to mediate endosomal and lysosomal PtdIns3P and mildly affect autophagy initiation. WDR65 also inhibited early autophagy events. However, the detailed mechanism for these proteins are yet to be determined. In summary, our work provided more understanding on the egulation of autophagosome formation, as well as a list of potentially novel regulators.