The application of molecular targeted therapies is expected to cause a modulation of cellular signaling pathway(s) that can be monitored by sequential biopsies. Fine-needle sampling (FNS) is an atraumatic and safe technique that can be repeated at numerous points during the clinical or experimental administration of a drug. However, small volume and paucicellularity of fine-needle samples may preclude a comprehensive analysis. We describe here the image-based detection of phosphorylated signaling proteins, an approach for the measurement of pathway activities and preliminary concepts for a multiplexed analysis in these specimens. Fine-needle samples were obtained from xenograft tumors and used for cell block preparations. Preanalytical parameters for the detection of phosphorylated Stat3 and nuclear factor kappaB were determined. A cytometric approach for the measurement of pathway activities was tested using 2 different slide-based analysis techniques applied to immunofluorescence and immunohistochemistry. Changes in the phosphorylation state of Stat3 and nuclear factor kappaB were observed due to delayed fixation and reproducibly quantified. Data obtained from xenografts after drug treatment suggest that slide-based cytometry gives results that are comparable to conventional analysis methods. The applicability of quantum dot nanocrystals for the detection of phosphorylated Stat3 and the combination of different labeling techniques suggest a potential for a multiplexed analysis. We propose here that FNS of solid tumors may be useful in anatomic sites where core-needle biopsies are not possible or not well tolerated. FNS can be used for biomarkers with a homogeneous distribution throughout the tumor, and slide-based analysis techniques may be applied to quantify pathway activities.