In the context of climate change, the stable isotope analysis of tree rings may play a crucial role in deciphering the eco-physiological mechanisms underlying forest decline and dieback phenomena. However, this technique is often considered expensive, time-consuming, and with several methodological constraints. Specifically, milling and transferring the material from jars to vials during the different steps of sample preparation involve risk of contamination among samples and loss of sample material. When dealing with declining trees (i.e., trees affected by loss of vitality with strong percentage of defoliation and reduction in growth) and trees subjected to extreme events or negative pointer years (characterized by extremely narrow ring) the sample preparation is particularly difficult because of scarce amount of wood material. In such a case, pooling rings from several years to achieve the minimum weight of wood is often necessary, thus losing information at the annual resolution. In order to overcome such limitations, we developed a novel protocol for quick and accurate whole-wood pre-processing, testing it on oak tree rings of different widths taken from living trees. The main novelty introduced by our protocol was freezing tree-ring samples at −80 °C and milling multiple samples at a time by using a 24-tube plate. The results showed that our novel simplified protocol significantly reduced the pre-processing time with respect to the standard protocol (12 vs. 284 sec/sample), while achieving the same wood particle size, limiting the loss of wood material and reducing the risk of contamination among samples.