Second harmonic imaging is a non-linear optical process that relies on the interaction of 2 photons with the sample. In this interaction, a photon of half the wavelength of the initial incoming light is generated. The generation of second harmonic signals requires the sample to have non-centrosymmetric molecular organization. Microtubules are composed of polymerized tubulin dimers and they are a major component in the cytoskeleton of neurons. Axons, in particular, have unipolarized microtubules with the plus ends growing away from the soma. This leads to an aligned microtubule organization in the axon, which allows second harmonic generation (SHG) and therefore label-free visualization of microtubule substructure (Dombeck et al. 2003, Stoothoff et al. 2008). Furthermore, it has been shown that microtubule alterations can be detected in rat and mouse primary neurons making use of SHG microscopy (Psilodimitrakopoulos et al., 2013, Van Steenbergen et al. 2019). The field of neuroscience has, so far, mostly relied on animal models to investigate neuronal physiology. The use of human induced pluripotent stem cell (iPSC)-derived neurons makes it possible to study neuronal physiology and health in human, disease-relevant genetic backgrounds. Here, we show that besides using fluorescence-based calcium imaging to monitor network activity, we can exploit the SHG signals to assess microtubule health in human iPSC-derived neurons.Figure 1: Live-cell, label-free imaging of microtubules of human iPSC-derived neurons