Intraluminal valves of collecting lymphatic vessels ensure unidirectional lymph transport against hydrostatic pressure gradient. Mouse mesentery harbors up to 800 valves and represents a convenient model for lymphatic valve quantification, high resolution imaging of different stages of valve development as well as for analysis of valve function. The protocol describes embryonic and postnatal mesenteric lymphatic vessel preparation for whole-mount immunofluorescent staining and visualization of valve organization, quantification of main morphological parameters such as valve size and leaflet length, and the quantitative assessment of functional properties of adult valves using back-leak and closure tests.