Abstract: In this study, we applied different fluorescent techniques to analyse the induction of apoptosis in the copepod Acartia tonsa nauplii exposed to low concentrations of nickel chloride (NiCl2). Newly hatched and later naupliar stages were exposed to increasing concentrations of NiCl2 (0.016, 0.025 and 0.063 mg·L−1) for 7 days and then stained with annexin V–FITC, a vital fluorescent probe commonly used to visualise apoptotic cells in live samples. Nauplii were also stained with TUNEL, a non-vital fluorescent probe used to detect apoptosis in fixed copepods. Moreover, we used for the first time, two-photon fluorescence (2PF) microscopy and higher harmonic generation microscopy (second SHG and third THG harmonic generation) to study apoptosis induction in A. tonsa nauplii without the use of fluorescent probes. 2PF and THG intensity increased in samples exposed to higher Ni concentrations, with respect to the control, whereas SHG signals were similar in all treated samples and visualised muscles. Future perspectives on the use of these new technologies to reveal apoptosis are discussed.