Assessment of biodegradability of carbon nanotubes (CNTs) is a critically important aspect that needs to be solved before their translation into new biomedical tools. CNT biodegradation has been shown both in vitro and in vivo, but we are limited by the number of analytical techniques that can be used to follow the entire process. Photothermal imaging (PhI) is an innovative technique that enables the quantitative detection of nanometer-sized absorptive objects. In this study, we demonstrate that PhI allows the observation of the degradation process of functionalized multi-walled carbon nanotubes (MWCNTs) following their internalization by primary glial cells. The absence of interference from the biological matrix components, together with the possibility to combine PhI with other detection techniques (e.g. fluorescence, light or electron microscopy) validate the potential of this method to follow the fate and behavior of carbon nanostructures in a biological environment.