Provided are methods and systems for reference object assisted droplet detection, indexing, and sorting in assays and diagnostics. Provided are compositions of reference objects and methods of usages. Provided are systems and exemplary modules and functions for the detection, counting, indexing, data processing, and sorting of reference objects and assay droplets in a microfluidic device. The reference objects may serve as a dynamic micro-scale positioning system for the indexing and sorting of individual assay droplets.