The present disclosure provides a membrane separation method of a cell suspension which can appropriately separate cells from debris, and a cell culture device. That is, membrane separation processing of the cell suspension is performed using a filtration membrane which includes an inlet-side opening formed on one surface and an outlet-side opening, which is formed on the other surface and communicates with the inlet-side opening, and in which the inlet-side opening and the outlet-side opening are disposed at positions deviated in a direction parallel to the surfaces of the membrane.