Spatially distributed chlorophyll content of urban vegetation provides an important indicator of a plant’shealth status, which might depend on the habitat quality of the specific urban environment. Recentadvances in optical remote sensing led to improved methodologies to monitor vegetation properties.The hyperspectral index NAOC (Normalized Area Over reflectance Curve) is one of these new tools thatcan be used for mapping chlorophyll content. In this paper we present the work done to quantify vege-tation chlorophyll content over the city of Valencia (Spain) based on chlorophyll measurements of fourrepresentative tree species: the London plane tree (Platanus x. acerifolia), the Canarian date palm (Phoenixcanariensis), the European nettle tree (Celtis australis) and the white mulberry (Morus alba). Measurementswere acquired during the summer of 2011, in a field campaign in which for 320 leaf samples, chlorophyllcontent was measured both in the laboratory and by using a SPAD-502 chlorophyll meter. Both methodswere correlated (R2> 0.86), using best fit power type functions. During the field campaign an aircraftwith a CASI (Compact Airborne Spectral Imager) hyperspectral sensor onboard overflew the city obtain-ing imagery with a spatial resolution of ∼1 m suitable to identify individual urban trees. From the CASIdata the NAOC index was calculated and linked with the laboratory chlorophyll content measurements.This led to a detailed chlorophyll content map with a RMSE of 15 g cm−2. Chlorophyll map analysis atthe individual crown level suggests the applicability to identify trees with lowered chlorophyll contentdue to a suboptimal habitat quality.