Functional organic materials with enhanced two-photon absorption lead to new technologies in the fields of chemistry, biology, and photonics. In this article we review experimental and theoretical methodologies allowing detailed investigation and analysis of two-photon absorption properties of organic chromophores. This includes femtosecond two-photon excited fluorescence experimental setups and quantum-chemical methodologies based on time-dependent density functional theory. We thoroughly analyze physical phenomena and trends leading to large two-photon absorption responses of a few series of model chromophores focusing on the effects of symmetric and asymmetric donor/acceptor substitution and branching.