Although in vivo and in vitro approaches appear to be very different, they are related and complementary techniques and both are essential for the investigation of diverse biological topics. The employment of both techniques was considered particularly appropriate to investigate the role of 17β-oestradiol and testosterone in echinoid reproductive biology. The relationship between sex-steroids and echinoid reproduction has not been clearly determined yet, due to the highly variable and sometimes contrasting results obtained from steroid administration experiments. These might be due to the activation of protective metabolic mechanisms that can prevent the exogenous molecules from exerting their biological functions, as observed in our previous research. To clarify these aspects, in the present study we explored sex-steroid involvement in the reproduction of the sea urchin Paracentrotus lividus, employing both in vivo and in vitro approaches: (1) an experiment involving hormone dietary administration was performed and different reproductive parameters were deeply analysed; (2) ovarian cells were cultured in the presence of the same steroids and morphological and biochemical analyses were carried out. According to our results, sex-steroids appear not to be involved in sea urchin gonad development and gamete maturation, as neither in vivo administration nor in vitro exposure influenced gonad and gamete growth. In addition, in vitro hormonal treatment did not affect sea urchin Major Yolk Protein content. Overall, the present work complements our previous research providing information on sex-steroid involvement in echinoid reproduction and illustrates new methodological approaches that will be useful for future research on invertebrate biology and physiology.