The imaging of tumors using techniques such as magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET), and near infrared fluorescence (NIRF) has seen significant advances in recent years. Two-photon fluorescence microscopy (2PFM) offers several advantages, such as deeper tissue penetration along with more confined excitation and emission that leads to an increase in imaging resolution. Although biomedical applications of two-photon fluorescence microscopy (2PFM) are steadily increasing, the technique still suffers from the lack of efficient, photostable two-photon absorbing fluorescent probes that possess high target specificity. In order to be truly useful for such applications, it is necessary to have not only an imaging component that undergoes two-photon absorption (2PA) at wavelengths longer than 700 nm, but also a vector that targets the fluorescent probe selectively to a particular tissue, cell, organelle, receptor, or protein. We describe approaches to 2PFM imaging that targets important biomarkers, including proteins that are expressed in tumors and the targeting of vasculature around the tumors rather than tumors themselves. In this work, we describe the preparation and the use of novel, efficient two-photon absorbing (2PA) fluorescent probes that target the vascular endothelial growth factor 2 (VEGFR-2), folate receptors, and integrin proteins. Comprehensive linear and nonlinear photophysical and biological characterization of the new probes will be presented along with 2PFM imaging.