Poly(β-malic acid) hydrophobic derivatives are promising polymers for biomedical and pharmaceutical applications. The objectives of the present work were to study the in vitro degradation profile of three PMLA hydrophobic derivatives and to evaluate their cytotoxicity before and after degradation. For this purpose, nanoparticles from poly(benzyl-malate) (PMLABe), poly(hexyl-malate) (PMLAHe), and poly-(malic acid-co-benzyl-malate) (PMLAH/He) were prepared for degradation studies on standardized materials. Size exclusion chromatography (SEC) and 1H NMR indicated that degradation occurred by random hydrolysis of the polymer main chain for all three polymer derivatives. The presence of carboxyl groups on the side chain and their esterification with different alcohols varying hydrophilicities could affect the degradation rate. It was postulated that the degradation depended on the rate of diffusion of water into the core of the particles. The cytotoxicity of the polymer nanospheres as well as their degradation products were evaluated in vitro with J774 A1 murine macrophage-like cell line. The cytotoxicity depended on the degradation rate of the polymers and the amount of degradation products of low molecular weight produced.