This review describes the development and some pilot applications of a new technology: 3D tissue image cytometry. 3D tissue image cytometry quantifies tissue morphology and biochemistry in a high throughput fashion. Promising applications range from cardiology, cancer biology, to tissue engineering. In this review, we will first examine traditional cytology, histology, and cytometry techniques that are the precursors of 3D tissue image cytometry. We will consider novel 3D optical imaging methods that form the technological basis for this new technology. Experiments for the validation and characterization of this instrument are presented. An application of this technology focusing on cardiac hypertrophy study is described in detail