This paper presents the results of the new interdisciplinary research done on Leonhard Rauwolf's herbarium with plants from the Middle East, which was later owned by Emperor Rudolf II. Using various sources, it examines how the herbarium came into the imperial collections, Early Modern methods of botanical research as described by Rauwolf in his printed travelogue, and how the illustrations for the printed book were produced from the specimens in the herbarium. The appendix (available in the online version) presents the new corrected botanical identification of the c. 200 plants in the fourth volume of Rauwolf's herbarium, and a correct transcription of the Early Modern Latin and vernacular names Rauwolf collected for these plants.