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Background Tumor-infiltrating lymphocytes (TILs) have yielded promising clinical results as an adoptive cell therapy for numerous solid tumors. Recently, TIL therapy received regulatory approval for melanoma, underscoring the need for robust, scalable product manufacturing and quality analysis. A persistent hurdle is the lack of rapid, contamination-free expansion workflows coupled with tools capable of resolving the functional state and clonotype makeup of the final cell product prior to infusion. Here, we present an integrated expansion-single cell sequencing strategy to address these gaps.Methods Dissociated lung tumor cells were loaded onto the CliniMACS Prodigy® system on which TILs were expanded for 14 days. High-throughput single cell analysis to generate whole transcriptome and T cell receptor (TCR) profiles using a combinatorial barcoding-based workflow was performed on an aliquot of the day 14 product. Illumina sequencing was performed and the data were processed with standard bioinformatic pipelines.Results The Prodigy workflow yielded expanded TILs in less than two weeks. Single cell whole transcriptome data identified all expected T cell subtypes and their cellular state. Further, TCR profiling recovered > 92% paired TCR-α/β CDR3 sequences, enabling confident clonotype assignment for nearly all profiled cells. Clonotype-frequency analysis identified hyperexpanded clones, revealing the existence of tumor-reactive T cells warranting further investigation.Conclusions Our workflow couples a rapid, closed-loop expansion system with single cell analysis to generate a scaled and molecularly annotated TIL product. The integration of transcriptome and clonotype data provided an immediate read out of both cell state and clonal make up of the TCR repertoire, critical information that could guide product release criteria or further engineering efforts. Indeed, by enabling faster, safer, and more precisely characterized TIL therapies, this workflow may enable immunologists to rapidly create safer, patient-specific treatments.