The diagnosis of growth hormone (GH) deficiency (GHD) is still problematic for the clinician. There is no gold standard for estimating GH secretion. The aim of this study was to compare the diagnostic usefulness of spontaneous GH secretion test, pharmacological tests with insulin, clonidine, L-dopa, and glucagon, and IGF-I measurement in GHD. We studied 180 prepubertal, short children. Predictive values were calculated for different GH cutoff levels for each diagnostic test. ROC curves were used to estimate the diagnostic usefulness of the tests. The results show that sleep is the strongest stimulatory agent for GH secretion. The estimation of GH secretion after onset of sleep can be used as a screening test in GHD diagnosis. The insulin test has the highest discrimination. A combination of insulin test with another provocative test allows high discrimination and accuracy for standard cut-off GH level. Measurement of IGF-I is characterized by low predictive values. IGF-I level below the mean according to age indicates high probability of GHD. Auxological parameters should be the most important factor in diagnosing GHD.