Certain probiotics can regulate the host's neurobehavioral function through the microbiota-gut-brain axis. However, screening these probiotics is mainly carried out in animal models, and is costly and inefficient. Herein, a putative enterochromaffin cell line (RIN14B) was used as an in vitro pre-screening model; 30 bacterial strains were tested for bacteria-stimulated tryptophan hydroxylase 1 gene (Tph1) expression and 5-hydroxytryptophan/5-hydroxytryptamine secretion. All strains were further validated for their neurobehavioral effects in chronic stress-induced depressive mice. Using partial least squares (PLS) modeling of in vitro and in vivo datasets, we found that the level of Tph1 mRNA in RIN14B significantly correlated with the performance of a forced swim test and sucrose preference test, and serum corticosterone level in chronically stressed mice. Four strains were identified as the best candidates among 30 strains using principal component analysis on all in vivo measures, and unsurprisingly, three of them could enhance Tph1 expression in RIN14B, which further proved that the RIN14B-based screening method (especially the detection of bacteria-stimulated Tph1 mRNA) has good predictive validity and screening efficiency for the strain's antidepressant-like capacity. Collectively, this study provides a novel in vitro method for screening probiotics (or other related bioproducts) with antidepressant-like potential.