Lipophilicity is a very important property while designing new drugs. Its influence on the pharmacological responses of compounds has been known since the 19th century. Also, many researchers try to find the relationships between structure and activity of newly synthetized compounds. The aim of this study was to determine the lipophilicity of 5,8-quinolinedione derivatives using thin-layer chromatography. Moreover, the structure—activity correlation between experimental and theoretical parameters of lipophilicity and anticancer activity was described. A series of 18 compounds was investigated. Experimental lipophilicity was determined by use of reversed-phase thin-layer chromatography (RP-TLC) using RP-18 F254s plates impregnated with silicone oil and the mixture of acetone and water solution of buffer Tris (pH = 7.4) in different volume compositions as mobile phases. Values of calculated lipophilicity (ALOGPs, AClogP, miLogP, KOWWIN, XLOGP2, MLogP, ABlogP, and ACD/LogP) were taken from the internet database. The correlation between experimental and calculated values of lipophilicity was found. For some compounds investigated, the calculated values of lipophilicity were relatively high. The correlation coefficient for these relationships was not very high but statistically significant. It confirms that the structure of compounds investigated affects the value of lipophilicity. The correlation between lipophilicity and anticancer activity was also determined, but unfortunately, no correlation was found. Additionally, similarity analysis was prepared for compounds investigated and results of lipophilicity were obtained.