The main goal of this study was the assessment of the N, N-disubstituted-2-chloroacetamides’ biological profile by chromatographic and in silico approach. Preliminary, their drug-like as well as lead-like properties and the bioactivity scores were analyzed. By applying reversed phase thin-layer chromatography as the simulator of compound’s distribution across biological membranes, the assumed bioactivity descriptors of the studied chloroacetamides were obtained. The compliance between obtained chromatographic parameters (R M,0 m) and relevant software lipophilicity/pharmacokinetic/ecotoxicity predictors of studied derivatives were examined by linear regression and additionally supported by more detailed relations established by multivariate methods. Also, it was confirmed that the studied bioactivity properties of the given chloroacetamides are conditioned by the type of the present hydrocarbon substituents (number, arrangement and binding of C atoms).