The relationship of the structure of polymers for targeted tumor therapy and their body fate in vivo is investigated within this work. Three different polymer classes have been characterized in a mouse model by use of non-invasive near-infrared multispectral fluorescence imaging: HPMA copolymers, carbohydrates and polyglutamates. The biodistribution pattern in vivo particularly depended on the polymer class. Nonetheless, a tumor accumulation in human colorectal carcinoma xenografts has been demonstrated for all investigated polymers in a mouse model. Using two fluorescence dyes varying in the emission spectra, multispectral fluorescence imaging allowed simultaneous observation of the biodistribution of the polymers and a cleavable drug model. The promising results of the tumor accumulation studies ultimately led to a treatment study in tumor bearing mice with a doxorubicin-conjugated HPMA copolymer. In this study, an improved efficacy and reduced toxicity of the polymer-drug conjugate compared to free doxorubicin has been demonstrated, which emphasizes the potential of polymers conjugates as a drug delivery system in tumor therapy.