A continuous monitoring of the whole tumor burden of individuals in orthotopic tumor models is a desirable aim and requires non-invasive imaging methods. Here we investigated whether quantification of a xenograft tumor intrinsic fluorescence signal can be used to evaluate tumor growth and response to chemotherapy. Stably fluorescence protein (FP) expressing cell clones of colorectal carcinoma and germ cell tumor lines were generated by lentiviral transduction using the FPs eGFP, dsRed2, TurboFP635, and mPlum. Applying subcutaneous tumor models in different experimental designs, specific correlations between measured total fluorescence intensity (FI) and the tumor volume (V) could be established. The accuracy of correlation of FI and V varied depending on the cell model used. The application of deep-red FP expressing xenografts (TurboFP635, mPlum) was observed to result in improved correlations. This was also reflected by the results of a performed error analysis. In a model of visceral growing mPlum tumors, measurements of FI could be used to follow growth and response to chemotherapy. However, in some cases final necropsy revealed the existence of additional, deeper located tumors that had not been detected in vivo by their mPlum signal. Consistently, only the weights of the tumors that were detected in vivo based on their mPlum signal correlated with FI. In conclusion, as long as tumors are visualized by their fluorescence signal the FI can be used to evaluate tumor burden. Deep-red FPs are more suitable for in vivo applications as compared to eGFP and dsRed2.