This study presents the results of the technical investigation carried out on several English portrait miniatures painted in the 16th and 17th century by Nicholas Hilliard and Isaac Oliver, two of the most famous limners working at the Tudor and Stuart courts. The 23 objects chosen for the analysis, spanning almost the entire career of the two artists, belong to the collections of the Victoria and Albert Museum (London) and the Fitzwilliam Museum (Cambridge). A non-invasive scientific methodology, comprising of stereo and optical microscopies, Raman microscopy, and X-ray fluorescence spectroscopy, was required for the investigation of these small-scale and fragile objects. The palettes and working techniques of the two artists were characterised, focusing in particular on the examination of flesh tones, mouths, and eyes. These findings were also compared to the information written in the treatises on miniature painting circulating during the artists’ lifetime. By identifying the materials and techniques most widely employed by the two artists, this study provides information about similarities and differences in their working methods, which can help to understand their artistic practice as well as contribute to matters of attribution.