Coral reefs are among the most diverse and iconic ecosystems. Over recent decades, coral reefs have declined significantly due to climate change resulting in an increased frequency and severity of summer heat waves. Corals live in symbiosis with single-celled microalgae, which play a vital role in providing their host with nutrients and also influence their heat tolerance. Hybridisation is a promising approach for the generation of novel microalgae geno and phenotypes. This study aimed to optimise the hybridisation of microalgae via protoplast cell fusion. Here, microalgae protoplasts were successfully produced through enzymatic digestion of their cell wall. This was validated by confocal microscopy analyses and flow cytometry. In addition, microalgae hybridisation was trialled with fusing agents and tracked using intracellular dyes. This proof-of-concept method enables experimentation with both inter and intraspecific hybridisation, paving the way for novel approaches to study the thermal tolerance mechanisms of symbiotic microalgae and potential contribution to innovative intervention techniques.