In the field of reproductive biology, there is a strong need for a suitable tool capable of non-destructive evaluation of oocyte viability and function. We studied the application of brilliant cresyl blue (BCB) as an intra-vital exogenous contrast agent using multispectral optoacoustic tomography (MSOT) for visualization of porcine ovarian follicles. The technique provided excellent molecular sensitivity, enabling the selection of competent oocytes without disrupting the follicles. We further conducted in vitro embryo culture, molecular analysis (real-time and reverse transcriptase polymerase chain reaction) and DNA fragmentation analysis to comprehensively establish the safety of BCB-enhanced MSOT imaging in monitoring oocyte viability. Overall, the experimental results suggest that the method offers a significant advance in the use of contrast agents and molecular imaging for reproductive studies. Our technique improves the accurate prediction of ovarian reserve significantly and, once standardized for in vivo imaging, could provide an effective tool for clinical infertility management.