Designing therapeutic vaccines has been a major focus in cancer immunotherapy. We have developed Galsomes, a cancer vaccine consisting of a lipid nanoparticle that incorporates tumor antigen mRNA and the glycolipid -galactosylceramide. Consequently, vaccination with Galsomes activates two types of adaptive and innate immune killer cells: cytotoxic T lymphocytes (CTLs), natural killer T (NKT) cells, respectively. To fully capitalize on these cells’ ability to eradicate cancer cells, it is critical to understand which barriers within the tumor-bearing host might form obstacles for their activity. Programmed death-ligand 1 (PD-L1) is such a barrier that can act on CTLs and NKT cells both at the time of activation as at the time of their effector function. There are indications that Galsome vaccination triggers expression of PD-L1, but up to now, no detailed information on the timing and location of this checkpoint expression is available. We previously developed a nanobody-mediated strategy to noninvasively image PD-L1. In this study, we used this nanobody-mediated SPECT/CT imaging strategy to determine the spatio-temporal expression of PD-L1 upon Galsome vaccination in the B16-OVA mouse melanoma tumor model. Moreover, ex vivo analysis was performed to support the SPECT/CT images. We also studied how Galsome vaccination combined with PD-L1 blockade impacts on the therapy outcome. Noninvasive, nanobody-mediated imaging of PD-L1 and ex vivo analysis was performed at several time points after Galsome vaccination in B16-OVA bearing mice, showing upregulation of PD-L1 expression as soon as one day after vaccination in organs targeted by the vaccine (lung, spleen, lymph node), while PD-L1 upregulation in the tumor environment occurred at later time points. Corroborating these findings, we showed that Galsome vaccination combined with anti-PD-L1 monoclonal antibody therapy significantly improved therapy outcome. In conclusion, Galsome vaccination in combination with PD-L1 blockade represents a promising, more effective treatment regimen for melanoma, a treatment regimen that is supported by noninvasive imaging of PD-L1 upon Galsome vaccination.