In the present study, we demonstrated the timing-controlled division dynamics of DNA droplet-based artificial cells by coupling them with non-equilibrium chemical reactions, resulting in the pathway control of artificial cell division (Figure ). We used DNA droplets constructed by mixing two Y-shaped branched DNA nanostructures (YA and YB; called binary-mixed DNA droplets), in which 6-branched DNA linkers crosslinked YA and YB (Figures and). Mixed DNA droplets are divided into YA and YB by cleaving the DNA linkers through the hybridization with division trigger DNAs. Here, we coupled the mixed DNA droplet with non-equilibrium chemical reactions; the time delay of division triggers (Figure ) realized timing and pathway control of DNA droplet division (Figures ). We used temporal control of DNA reactions based on RNA degradation with a ribonuclease H (RNase H), which has been used in many dynamic DNA reactions such as DNA oscillators , DNA bistable switch , logic computation 50 , DNA walker , and timers for DNA strand displacement reactions ; however, there is no report on temporal control of LLPS droplets with the RNase H reaction. Finally, we present a molecular computing element to compare the concentrations of microRNA (miRNA) sequences (called molecular comparators) as an application of the timing-controlled division of DNA-droplet-based artificial cells. Our results provide a method for chemically regulating the timing-controlled physical dynamics of LLPS droplets for artificial cell studies.