A sensitive high performance liquid chromatographic method has been developed and validated for the determination of proparacaine in human aqueous humour. The procedure involved extraction of proparacaine from aqueous humour with cyclohexane. The separation was achieved using a Bondesil C-8 (250 x 4.6 mm i.d., particle size 5 mu m) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH 3.0, 20 mM) (30:70, v/v). Proparacaine and liclocaine (internal standard, IS) detection was performed by UV-Vis detector at 220 nm. The retention times for proparaca ine and IS were 12.01 and 5.58 min, respectively. HPLC-UV-Vis method was linear in the range of 75-4,000 ng mL(-1). The limit of detection (LOD) was 25 ng mL(-1) and the limit of quantification (LOQ) of proparacaine was found to be 75 ng mL(-1) (RSD <= 15%, n = 6). In intra- and inter-day precision and accuracy analysis, the relative standard deviation was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Recovery of proparacaine from human aqueous humour was 99.98% at 500 ng mL(-1). Proparacaine solutions were stable at least 6 months at +4 and -20 degrees C. Proparacaine levels of aqueous humour in fifteen volunteers' were in the range of 80.21 and 459.00 ng mL(-1). According to system suitability tests and Shewhart's quality control charts the proparacaine responses were in the acceptance ranges. Developed method was providing a sufficient quality at least over 3 months for determination of proparacaine in human aqueous humour.