In this work, we introduce a free and open-source Python package, coined ROSHAMBO (Rapid, Open Source, Helpful Application for Measuring Bioactive Overlapsgithub.com /molecularinformatics/roshambo), specifically designed for molecular alignment and 3D similarity calculations. Optimized for large-scale virtual screening of small molecules, this tool employs GPU accelerated alignment of molecular shapes using Gaussian overlays via PAPER 25 in the backend. Key features of ROSHAMBO include a user-friendly interface, optional conformer generation, the capability to compute hybrid similarity metrics (incorporating both shape and pharmacophore features), and a high degree of flexibility and customization. Its modular design facilitates easy integration into various research workflows, easily benchmarked through built-in analysis and visualization tools. Previous work compared ROCS and PAPER, ROSHAMBO's backend overlay optimization software, finding similar active compound enrichment on the DUD screening set. In this work, we further validate ROSHAMBO against a common open-source 3D shape similarity tool, showing superiority in terms of both speed and performance. ROSHAMBO thus addresses the current gaps in 3D molecular similarity analysis, providing a validated, accessible, and flexible opensource tool for use in myriad drug discovery workflows.