Ray parenchyma is an essential tissue for tree functioning and survival. This living tissue plays a major role for storage and transport of water, nutrients and non-structural carbohydrates (NSC), thus regulating xylem hydraulics and growth. However, despite the importance of rays for tree carbon and water relations, methodological challenges hamper knowledge about ray intra- and inter-tree variability and its ecological meaning. In this study we provide a methodological toolbox for soundly quantifying spatial and temporal variability of different ray features.Anatomical ray features were surveyed in different cutting planes (cross-sectional, tangential, and radial) using quantitative image analysis on stem-wood micro-sections sampled from 41 mature Scots pines (Pinus sylvestris). The percentage of ray surface (PERPAR), a proxy for ray volume, was compared among cutting planes and between early- and latewood to assess measurement-induced variability. Different tangential ray metrics were correlated to assess their similarities. The accuracy of cross-sectional and tangential measurements for PERPAR estimates as a function of number of samples and the measured wood surface was assessed using bootstrapping statistical technique. Tangential sections offered the best 3D insight of ray integration into the xylem and provided the most accurate estimates of PERPAR, with 10 samples of 4 mm2 showing an estimate within ±6.0% of the true mean PERPAR (relative 95% confidence interval, CI95), and 20 samples of 4 mm2 showing a CI95 of ±4.3%. Cross-sections were most efficient for establishment of time series, and facilitated comparisons with other widely used xylem anatomical features. Earlywood had significantly lower PERPAR (5.77 vs. 6.18%) and marginally fewer initiating rays than latewood. In comparison to tangential sections, PERPAR was systematically overestimated (6.50 vs. 4.92%) and required approximately twice the sample area for similar accuracy. Radial cuttings provided the least accurate PERPAR estimates. This evaluation of ray parenchyma in conifers and the presented guidelines regarding data accuracy as a function of measured wood surface and number of samples represent an important methodological reference for ray quantification, which will ultimately improve the understanding of the fundamental role of ray parenchyma tissue for the performance and survival of trees growing in stressed environments.