Context: Three-dimensional tumor spheroid cultures are a better representative of in vivo solid tumors than monolayer cultures and should be used for testing potential nanotherapeutics in vitro.Objective: To develop techniques to test the disposition and efficacy of nanocarrier formulations in spheroids in a cost-effective manner amenable to high-throughput testing.Methods: Spheroids were obtained using a modified liquid overlay technique in a 96-well plate. Several nanocarrier formulations were prepared and tested in the spheroid model. The disposition of the formulations in the spheroids was determined by confocal microscopy while the effect of the drug-loaded formulations was assessed in terms of the cell viability, loss of membrane integrity, induction of caspases and inhibition of growth of the spheroids.Results: The surface charge of the formulations influenced the accumulation of the nanocarrier and drug in the spheroid, with the cationic formulation accumulating to the greatest extent. Also, the smallest particle size formulation, micelles, penetrated to the greatest extent in the spheroid. The iRGD tumor-penetrating peptide co-administered with unmodified liposomes exhibited both high accumulation and penetration. The effect studies revealed that the formulations that penetrated or accumulated to the highest extent in the spheroid exhibited better antitumor activity compared to the other formulations.Conclusion: The 96-well plate format spheroid model developed in the study can be used toward the rational selection of nanocarrier therapeutics prior to their testing in in vivo models.