To quantify the domain localization phenomenon, we selected two specific lipid compositions leading to different Lo/Ld ratios, referred to as Comp.1 (DPPC/DOPC/Chol = 45/25/30 mol%) and Comp.2 (DPPC/DOPC/Chol = 30/45/25 mol%). Using fluorescence microscopy images, we first measured the projected area fractions of Lo and Ld phases in hundreds of GUVs before confinement and established how GUVs distributed according to their Ld fraction (Figure ). As expected, we found that the small fraction of unsaturated DOPC lipid in Comp.1 led to a majority of Lo-rich GUVs while the DOPC-rich Comp.2 resulted in a majority of Ld-rich GUVs. Without confinement, this phase separation was accompanied by domain coalescence and budding of the phase in minority, i.e., Ld and Lo for Comp.1 and Comp.2, respectively (Figure , top). Interestingly, when phase separation occurred in the confined state, we observed that lipid domains corresponding to the minority phase were mainly localized in the free curved regions resulting in rod-like shaped GUVs with those domains at their tips (Figure , bottom), in agreement with the previous time-lapse observation (Figure ). Hence, with Comp.1 (resp. Comp.2), Lo-rich (resp. Ld-rich) GUVs had Ld (resp. Lo) domains localized at their tips. We thus obtained the same patterning (minority phase at tips and majority phase in between) with opposite distribution of Lo and Ld phase depending on the initial lipid composition. To characterize the extent of domain localization achieved with this method, we established the distribution of Ld phase, as the projected red fluorescence on the microscopy images, along a normalized longitudinal axis of each confined GUV divided into 20 areas (Figure , top). We repeated this analysis on a large number of confined GUVs (Figure ) and plotted the median value of Ld ratio along this axis for different compositions and geometries of confinement (Figure , bottom). This analysis demonstrates a strong and systematic domain localization for both compositions, with Ld domains localizing at the tip (free region) of Lo-rich GUVs or in the middle body (confined part) of Ld-rich GUVs. The decrease in the Ld ratio observed in the first and last 5% of the long axis of the confined Lo-rich GUVs was attributed to the small fraction of Lo phase that could be trapped there and which was overestimated by our segmentation analysis in this membrane curved region. Since complete switching of the phase localizing at the GUV free region was possible, the localization did not depend on the chemical nature of lipids nor on the type of phase, but was instead directed by the Lo/Ld phase ratio. For Comp.1, we also obtained similar distributions regardless of the width and height of the confining channel (Figure , bottom). This is attributed to a too low curvature at the free region to induce the type of domain sorting that could be observed with highly curved membranes. It also highlights the robustness of the approach to direct domain localization. All these results show that the minority-phase domains reproducibly accumulated, coalesced and grew at the free regions of the confined GUVs, regardless of the lipid nature and degree of confinement. This process is attributed to the minimization of line tension though domain accumulation, fusion and unconstrained membrane deformation in the free curved regions.