Growth is an important biological trait and monitoring metric for the assessment of the success and progress of restoration projects with the European oyster (Ostrea edulis). However, sampling time and frequency are often limited, as well as the ability to survey individual growth. Chemical dyes, such as calcein, can be used to create incremental markings for measuring growth in shell cross-sections, especially in bivalves. However, potential negative effects and limits for successful staining have not been assessed for O. edulis, yet. In this study, three different calcein concentrations (100 mg l⁻¹, 150 mg l⁻¹, 200 mg l⁻¹) with three different immersion times (6 h, 12 h, 24 h) were tested for potential negative effects, the best incremental marking result and their potential to be used in growth measurements. Furthermore, the deposition of an annual growth line was investigated. Results showed that calcein is a reliable in situ fluorescence marker that produced sufficient growth lines in the cross-section of O. edulis. At a concentration of 100 mg l⁻¹ or higher and immersion times of 6 h or more, no negative effects on growth and survival were observed after five months. Applications include the possibility for long-term, individual growth data for a large number of oysters for restoration monitoring as well as the option of marking restored oysters from aquaculture production to distinguish them from existing wild stock populations or natural offspring.