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In single-cell RNA-seq (scRNA-seq) data analysis, a fundamental problem is to determine the number of cell clusters based on the gene expression profiles. However, the performance of current methods is still far from satisfactory, presumably due to their limitations in capturing the expression variability among cell clusters. Batch effects represent the undesired variability between data measured in different batches. When data are obtained from different labs or protocols batch effects occur. Motivated by the practice of batch effect removal, we considered cell clusters as batches. We hypothesized that the number of cell clusters (i.e. batches) could be correctly determined if the variances among clusters (i.e. batch effects) were removed. We developed a new method, namely, removal of batch effect and testing (REBET), for determining the number of cell clusters. In this method, cells are first partitioned into k clusters. Second, the batch effects among these k clusters are then removed. Third, the quality of batch effect removal is evaluated with the average range of normalized mutual information (ARNMI), which measures how uniformly the cells with batch-effects-removal are mixed. By testing a range of k values, the k value that corresponds to the lowest ARNMI is determined to be the optimal number of clusters. We compared REBET with state-of-the-art methods on 32 simulated datasets and 14 published scRNA-seq datasets. The results show that REBET can accurately and robustly estimate the number of cell clusters and outperform existing methods. Contact: H.D.L. (hongdong@csu.edu.cn) or Q.S.X. (qsxu@csu.edu.cn) [ABSTRACT FROM AUTHOR]