We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.