Aqueous humor (AH) is a transparent fluid found in the anterior chamber of the eye. The circulating AH nourishes the cornea and lens and removes the metabolic waste moving through the ocular chambers and drains from the eye to the venous blood. Analysis of drugs in AH is necessary to evaluate their pharmacokinetics parameters, which may be crucial to avoid potential adverse effects. Analysis of endogenous components of AH may help to understand its physiology as well as changes evoked by pathological situation. This review describes analytical methods used for determination of pharmaceuticals and small endogenous molecules in AH, focusing on sample preparation procedures and analytical techniques. Studies on human and animal samples are included. After inspection and filtering of records found in PubMed about 100 research papers were selected to review. In these articles AH samples of human and rabbit origin were studied most often. Sample evaporation and reconstitution in smaller solvent volume was the most popular method for analyte pre-concentration. Acetonitrile, methanol or mixture of both solvents were used most often for protein precipitation.