A novel strategy for the direct analysis of non-conjugated steroids in water using paper spray mass spectrometry (PS-MS) has been developed. PS-MS was used in the identification and quantification of non-conjugated (free) steroids in fish tank water samples. Data shown herein indicates that individual amounts of free steroids can be detected in aqua as low as; 0.17 ng/µL, 0.039 ng/µL, 0.43 ng/µL, 0.0076 ng/µL for aldosterone, corticosterone, cortisol, and β-estrone, respectively, and with an average relative standard deviation of ca. < 10% in the positive ion mode using PS-MS/MS. Direct detection of free steroids in a raw water mixture, from aquaculture, without prior sample preparation is demonstrated. The presence of free steroids released in fish water samples was confirmed via tandem mass spectrometry using collision-induced dissociation. This approach shows promise for rapid and direct water quality monitoring to provide a holistic assessment of non-conjugated steroids in aqua.