Abstract Body (Do not enter title and authors here): Plasma membrane integrity is vital for nearly all aspects of cell functioning. Mechanical forces can cause plasma membrane damage, but it is not known whether there are large molecules that regulate plasma membrane integrity under mechanical strain. Here we constructed a novel high-throughput cellular stretch system, and screened 10,843 siRNAs targeting 2,726 multi-pass transmembrane proteins for strain-induced membrane permeability changes. The screen identified NINJ1, a protein recently proposed to regulate pyroptosis and other lytic cell death in monocyte-like cells as the top hit. We demonstrate that NINJ1 is a critical regulator for mechanical strain-induced plasma membrane rupture (PMR) in circulating immune cells. NINJ1 level on the cell membrane is inversely correlated to the amount of force required to rupture the membrane. In the pyroptosis context, NINJ1 on its own is not sufficient to fully rupture the membrane, and additional mechanical force is required for full PMR. Our work establishes that NINJ1 functions as a bona fide determinant of membrane biomechanical properties. Our study also suggests that inflammation-induced PMR of circulating immunce cells is subjected to fine tuning by differences in NINJ1 expression and mechanical cues from microenvironments.