Follicles are isolated from ovaries for numerous reasons, including IVM, but adult murine yields are <2 folliclesmg-1. The aim of the present study was to optimise ovarian disaggregation and develop methods applicable to the rapid screening of follicle viability. Ovaries from adult mice (n=7) were halved and disaggregated mechanically, or by using collagenase IV (Col-IV; 590UmL-1) or animal origin-free collagenase IV (AOF) at 590 or 1180UmL-1. Isolated follicles were stained with 4',6'-diamidino-2-phenylindole (DAPI; nuclei), chloromethyl-X-rosamine (CMXRos; mitochondria) or fluorescein isothiocyanate-conjugated anti-α-tubulin antibody. Follicle diameters and staining were measured and analysed using ImageJ, and data analysed using GraphPad Prism. Col-IV disaggregation yielded the highest number of follicles (17±10 folliclesmg-1 ovarian tissue). All disaggregation methods released more secondary follicles (86±20 per ovary; P<0.05) than any other size cohort. Mechanical and Col-IV disaggregation yielded similar numbers of morphologically intact follicles, whereas AOF disaggregation caused more damage (P<0.01). As the morphological disruption increased, DAPI and CMXRos staining decreased (P<0.05), and tubulin localisation became more heterogeneous. Col-IV disaggregation gave the best yield of morphologically intact follicles containing viable granulosa cells. In conclusion, we improved adult murine follicle yields and applied molecular markers to assess follicle morphology, cellular cytoskeleton and mitochondrial function.