ABSTRACT Owing to their high efficacy, antimitotic chemotherapeutics are the mainstay for most cancer treatments. However, these drugs do not discriminate between tumor and healthy cells, thus show dose-limiting toxicity and severe adverse effects. To improve treatments, rendering chemotherapeutics tumor-cell specific is highly desirable. Although various strategies, such as targeted antibody–drug conjugates, aim to achieve this goal, the identification of a tumor-specific ‘Achilles' heel’ remains a challenge. Here, we followed an alternative approach, which does not rely on tumor-specific characteristics, but rather uses spatially confined illumination of the light-activatable microtubule inhibitor SBTubA4P to target its cytotoxic activity to tumor cells. We demonstrate that localized illumination of SBTubA4P allows for precise eradication of disseminated sarcoma cells in zebrafish xenografts without inducing systemic toxicity. In addition to the already-described light-dependent inhibition of microtubule dynamics by SBTubA4P, our data indicate that this molecule creates reactive oxygen species upon UV illumination, which significantly increases its cytotoxic effects. SBTubA4P is a valuable addition to the precision oncology toolbox, and zebrafish xenografts constitute a well-suited model to investigate photoactivatable compounds in vivo.