Background Non-invasive in vivo imaging strategies are of high demand for longitudinal monitoring of inflammation during disease progression. In this study we present an imaging approach using near infrared fluorescence (NIRF) imaging in combination with a polyanionic macromolecular conjugate as a dedicated probe, known to target L- and P-selectin and C3/C5 complement factors. Methodology/Principal Findings We investigated the suitability of dendritic polyglycerol sulfates (dPGS), conjugated with a hydrophilic version of the indocyanine green label with 6 sulfonate groups (6S-ICG) to monitor sites of inflammation using an experimental mouse model of allergic asthma. Accumulation of the NIRF-conjugated dPGS (dPGS-NIRF) in the inflamed lungs was analyzed in and ex vivo in comparison with the free NIRF dye using optical imaging. Commercially available smart probes activated by matrix metalloproteinase's (MMP) and cathepsins were used as a comparative control. The fluorescence intensity ratio between lung areas of asthmatic and healthy mice was four times higher for the dPGS in comparison to the free dye in vivo at four hrs post intravenous administration. No significant difference in fluorescence intensity between healthy and asthmatic mice was observed 24 hrs post injection for dPGS-NIRF. At this time point ex-vivo scans of asthmatic mice confirmed that the fluorescence within the lungs was reduced to approximately 30% of the intensity observed at 4 hrs post injection. Conclusions/Significance Compared with smart-probes resulting in a high fluorescence level at 24 hrs post injection optical imaging with dPGS-NIRF conjugates is characterized by fast uptake of the probe at inflammatory sites and represents a novel approach to monitor lung inflammation as demonstrated in mice with allergic asthma.