This paper presents the first extensive investigation of the composition of both the organic and inorganic media used by Lorenzo Lotto, one of the outstanding artists of the Italian Renaissance in the early 16th century. By investigating four paintings from The State Hermitage Museum collection (Saint-Petersburg, Russia), new insights into the artist’s painting techniques were obtained, which contribute to our understanding of the transition from egg tempera to oil painting techniques that took place in Italian paintings between the 15th to the mid 16th century. Inorganic materials were determined using SEM-EDX and PLM, revealing information on the artist’s palette. Of particular note was the powdered colourless glass found in the priming layer of the Madonna delle Grazie, which was most likely used as dryer. Organic materials were analysed in individual paint layers using GC-MS, and revealed a mature use of drying oils as paint binders, skilfully mixed with other organic materials and inorganic driers, to obtain the desired aesthetical and technical qualities of the paint. The study revealed that Lotto used the tempera grassa technique through most of his creative life.