In this paper, we present a centrifugal microfluidic concept employing event-triggered valving for automated extraction of metered plasma and peripheral blood mononuclear cells (PBMCs). This “lab-on-a-disk” system has been developed for retrieving different density layers from a liquid column by “overflowing” the layers sequentially using the pressure exerted by a density-gradient liquid. Defined volumes of plasma and PBMCs were efficiently forwarded into designated microfluidic chambers as a sample preparation step prior to further downstream processing. Furthermore, the extracted PBMCs were counted directly on-disk using an automated optical unit by object-based image analysis, thus eliminating the requirement for the post-processing of the extracted PBMCs. This study is a direct continuation of our previous work1 where we demonstrated combined on-disk detection of C-reactive protein and quantification of PBMCs following on-disk extraction of plasma and PBMCs from a single blood sample using a centrifugo-pneumatic valving mechanism. However, the former valving technique featured limited PBMC extraction efficiency. Here, integrating the novel concept along with event-triggered valving mechanism, we eliminated the occurrence of a specific microfluidic effect, which led us to increase PBMC extraction efficiency to 88%. This extraction method has the potential to be utilized for efficiently separating multiple density layers from a liquid sample in relevant biomedical applications.