Background: Several gene therapy strategies have been designed for cancer treatment. Intra‐tumoral injection of adenoviruses expressing pro‐drug‐converting enzymes is one such strategy. Although the efficacy of these therapies was tested in animal models, little work has been devoted to the determination of critical variables for success. In this work, we aimed at better understanding variables that affect the cytosine deaminase::uracil phosphoribosyl transferase (CD::UPRT)‐based strategy in two human glioblastomas. Methods: We have constructed two adenoviruses expressing either CD or the fusion protein CD::UPRT. We have tested their anti‐tumor activity in combination with 5‐fluorocytosine (5‐FC) in the glioblastoma cell lines U87 and U251, which are p53‐wt and p53‐deficient, respectively. Anti‐tumor activity has also been investigated in spheroid structures. Results: The superiority of CD::UPRT over CD was confirmed in both glioblastoma cells. We found that the pro‐drug concentration required for anti‐tumor activity was 9‐fold higher in U251 than in U87, while multiplicity of infection (MOI) as low as 6 was sufficient to achieve 50% killing. Bystander activity was observed with as few as 2 and 6% transduced cells for U87 and U251, respectively. Differences in sensitivity between U87 and U251 were not due to differences in transduction, transgene expression, or intercellular transport, but were related to 5‐FU sensitivity and p53 status. Also, it is noteworthy that, in contrast to U87, U251 spheroids barely responded to the treatment, whereas their monolayer counterparts were very sensitive. Conclusions: Our study has shown that p53 status is important for CD::UPRT/5‐FC treatment. Moreover, this study demonstrated that the three‐dimensional spheroid model is a more stringent in vitro model for suicide gene therapy evaluation than are monolayer cultures.