There are provided a processing method of a substrate in which in forming a trench on the substrate by etching, a side wall surrounding the trench is surely protected, and a manufacturing method of a liquid ejection head. The methods include: repeating sequentially a plurality of cycles of a trench forming step of forming the trench on a printing element substrate, a first protection layer forming step of forming a passivation layer, and a first protection layer removing step of removing a portion at which the trench is excavated in the passivation layer. A second protection layer forming step and a second protection layer removing step are performed between the trench forming step through the first protection layer removing step repeated in a plurality of cycles and the trench forming step through the first protection layer removing step repeated next.