Objectives Stable isotope analysis of sequential dentine samples is a potentially powerful method to reveal insights into early life-histories of individuals in the past. Dentine incremental growth structures are complex, however, and current approaches that apply horizontal sectioning of demineralized tooth halves or quarters risk combining multiple growth layers and may include unwanted cementum or secondary dentine. They also require destruction of large parts of a tooth. Here, we present a less destructive and relatively straightforward protocol that reduces damage, increases temporal resolution, and improves the accuracy of age-alignment between individuals. Material and methods We outline a protocol that includes the sampling of small (1 mm diameter) cylindrical plug transects from a thin section, along with an age-alignment scheme predicated on average growth rates for dentine areas. Results and discussion The proposed protocol is readily applicable and more anatomically sensitive than horizontal slicing. Micro-samples are smaller (in both length and depth), hence minimizing temporal overlap and avoid directions that may contravene growth pattern. They completely avoid areas where secondary and tertiary dentine or cementum can be deposited. Age-alignment is improved by using growth ratios of anatomical tooth zones. Conclusion This method minimizes destruction, enables finer temporal resolution and facilitates data comparison. It can be readily combined with fluorescence imaging-based or other pre-screening methods of dentine collagen preservation.