Motivation: Barcode sequencing (bar-seq) is a high-throughput, and cost effective method to assay large numbers of cell lineages or genotypes in complex cell pools. Because of its advantages, applications for bar-seq are quickly growing-from using neutral random barcodes to study the evolution of microbes or cancer, to using pseudo-barcodes, such as shRNAs or sgRNAs to simultaneously screen large numbers of cell perturbations. However, the computational pipelines for bar-seq clustering are not well developed. Available methods often yield a high frequency of under-clustering artifacts that result in spurious barcodes, or over-clustering artifacts that group distinct barcodes together. Here, we developed Bartender, an accurate clustering algorithm to detect barcodes and their abundances from raw next-generation sequencing data. Results: In contrast with existing methods that cluster based on sequence similarity alone, Bartender uses a modified two-sample proportion test that also considers cluster size. This modification results in higher accuracy and lower rates of under- and over-clustering artifacts. Additionally, Bartender includes unique molecular identifier handling and a 'multiple time point' mode that matches barcode clusters between different clustering runs for seamless handling of time course data. Bartender is a set of simple-to-use command line tools that can be performed on a laptop at comparable run times to existing methods. Availability and implementation: Bartender is available at no charge for non-commercial use at https://github.com/LaoZZZZZ/bartender-1.1. Contact: [email protected] or [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.