The 2D monolayer cell culture model is often the first step in the prediction of the success or failure of a nanoparticle-based drug delivery system. However, there is often poor translation between the 2D monolayer in vitro results and the nanoparticle-drug performance in vivo. One possible way of bridging this gap is the use of multicellular tumor spheroids (MCTSs) as an intermediate in vitro model due to its 3D structure. This paper aims to quantify and compare the results obtained from traditional 2D monolayer cell cultures and 3D MCTS by studying the cytotoxic effects of free paclitaxel (PTX) and paclitaxel, which has been conjugated to a poly(ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer and self-assembled to give a micellar delivery system. The core of the micelle was cross-linked with a diamino nondegradable cross-linker to compare the effects of micelle stability on the results. Although the 2D prostate tumor cell culture results indicated that all micellar variants (IC50: 193-271 nM) were significantly less toxic than free paclitaxel (IC50: 15.2 nM), the micelles showed faster and higher cytotoxicity than free PTX in the 3D prostate MCTS. The cross-linking of micelles even showed accelerated antitumor activities to the MCTS compared with un-cross-linked micelles. The results indicate that DAO-cross-linked POEGMEA-b-PCEA-PTX conjugate micelles will be a useful nanodrug carrier for prostate cancer therapy. MCTS offers a very promising method of incorporating 3D structures into in vitro testing.