The docking accuracy of Glide was evaluated using 16 different docking protocols on 190 protein-fragment complexes representing 78 targets. Standard precision docking (Glide SP) based protocols showed the best performance. The average root-mean-square deviation (rmsd) between the docked and cocrystallized poses achieved by Glide SP with pre- and postprocessing was 1.17 Å, and an acceptable binding mode with rmsd < 2 Å could be found in 80% of the cases. Comparison of the docking results produced by different protocols suggests that the sampling efficacy of Glide is adequate for fragment docking. The docking accuracy seems to be limited by the performance of scoring schemes, which is supported by the weak correlation between experimental binding affinities and GlideScores. Cross-docking experiments performed on 8 targets represented by 63 complexes revealed that Glide SP gave similar results to that of the computationally more intensive Glide XP. The average rmsd achieved by Glide SP with pre- and postprocessing was 2.06 Å, and an acceptable binding mode with rmsd < 2 Å could be found in 63% of the cases. These cross-docking results were improved significantly selecting the optimal X-ray structure for each target (average rmsd = 1.3 Å, success rate = 77%), indicating the importance of enrichment studies and the use of multiple X-ray structures in virtual fragment screening. © 2010 American Chemical Society.